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Sample records for dead box rna

  1. Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase.

    PubMed

    Theissen, Bettina; Karow, Anne R; Köhler, Jürgen; Gubaev, Airat; Klostermeier, Dagmar

    2008-01-15

    RNA helicases couple the energy from ATP hydrolysis with structural changes of their RNA substrates. DEAD box helicases form the largest class of RNA helicases and share a helicase core comprising two RecA-like domains. An opening and closing of the interdomain cleft during RNA unwinding has been postulated but not shown experimentally. Single-molecule FRET experiments with the Bacillus subtilis DEAD box helicase YxiN carrying donor and acceptor fluorophores on different sides of the interdomain cleft reveal an open helicase conformation in the absence of nucleotides, or in the presence of ATP, or ADP, or RNA. In the presence of ADP and RNA, the open conformation is retained. By contrast, cooperative binding of ATP and RNA leads to a compact helicase structure, proving that the ATP- and ADP-bound states of RNA helicases display substantially different structures only when the RNA substrate is bound. These results establish a closure of the interdomain cleft in the helicase core at the beginning of the unwinding reaction, and suggest a conserved mechanism of energy conversion among DEAD box helicases across kingdoms.

  2. RNA Remodeling Activity of DEAD Box Proteins Tuned by Protein Concentration, RNA Length, and ATP.

    PubMed

    Kim, Younghoon; Myong, Sua

    2016-09-01

    DEAD box RNA helicases play central roles in RNP biogenesis. We reported earlier that LAF-1, a DEAD box RNA helicase in C. elegans, dynamically interacts with RNA and that the interaction likely contributes to the fluidity of RNP droplets. Here we investigate the molecular basis of the interaction of RNA with LAF-1 and its human homolog, DDX3X. We show that both LAF-1 and DDX3X, at low concentrations, are monomers that induce tight compaction of single-stranded RNA. At high concentrations, the proteins are multimeric and dynamically interact with RNA in an RNA length-dependent manner. The dynamic LAF-1-RNA interaction stimulates RNA annealing activity. ATP adversely affects the RNA remodeling ability of LAF-1 by suppressing the affinity, dynamics, and annealing activity of LAF-1, suggesting that ATP may promote disassembly of the RNP complex. Based on our results, we postulate a plausible molecular mechanism underlying the dynamic equilibrium of the LAF-1 RNP complex. PMID:27546789

  3. Division of labor in an oligomer of the DEAD-box RNA helicase Ded1p

    PubMed Central

    Putnam, Andrea A.; Gao, Zhaofeng; Liu, Fei; Jia, Huijue; Yang, Quansheng

    2015-01-01

    Most aspects of RNA metabolism involve DEAD-box RNA helicases, enzymes that bind and remodel RNA and RNA-protein complexes in an ATP-dependent manner. Here we show that the DEAD-box helicase Ded1p oligomerizes in the cell and in vitro, and unwinds RNA as a trimer. Two protomers bind the single stranded region of RNA substrates and load a third protomer to the duplex, which then separates the strands. ATP utilization differs between the strand separating protomer and those bound to the single stranded region. Binding of the eukaryotic initiation factor 4G to Ded1p interferes with oligomerization and thereby modulates unwinding activity and RNA affinity of the helicase. Our data reveal a strict division of labor between the Ded1p protomers in the oligomer. This mode of oligomerization fundamentally differs from other helicases. Oligomerization represents a previously unappreciated level of regulation for DEAD-box helicase activities. PMID:26212457

  4. Division of Labor in an Oligomer of the DEAD-Box RNA Helicase Ded1p.

    PubMed

    Putnam, Andrea A; Gao, Zhaofeng; Liu, Fei; Jia, Huijue; Yang, Quansheng; Jankowsky, Eckhard

    2015-08-20

    Most aspects of RNA metabolism involve DEAD-box RNA helicases, enzymes that bind and remodel RNA and RNA-protein complexes in an ATP-dependent manner. Here we show that the DEAD-box helicase Ded1p oligomerizes in the cell and in vitro, and unwinds RNA as a trimer. Two protomers bind the single-stranded region of RNA substrates and load a third protomer to the duplex, which then separates the strands. ATP utilization differs between the strand-separating protomer and those bound to the single-stranded region. Binding of the eukaryotic initiation factor 4G to Ded1p interferes with oligomerization and thereby modulates unwinding activity and RNA affinity of the helicase. Our data reveal a strict division of labor between the Ded1p protomers in the oligomer. This mode of oligomerization fundamentally differs from other helicases. Oligomerization represents a previously unappreciated level of regulation for DEAD-box helicase activities.

  5. A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

    PubMed

    Ruminski, Dana J; Watson, Peter Y; Mahen, Elisabeth M; Fedor, Martha J

    2016-03-01

    RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo.

  6. A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

    PubMed

    Ruminski, Dana J; Watson, Peter Y; Mahen, Elisabeth M; Fedor, Martha J

    2016-03-01

    RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo. PMID:26759451

  7. DEAD/DExH-Box RNA Helicases in Selected Human Parasites.

    PubMed

    Marchat, Laurence A; Arzola-Rodríguez, Silvia I; Hernandez-de la Cruz, Olga; Lopez-Rosas, Itzel; Lopez-Camarillo, Cesar

    2015-10-01

    DEAD/DExH-box RNA helicases catalyze the folding and remodeling of RNA molecules in prokaryotic and eukaryotic cells, as well as in many viruses. They are characterized by the presence of the helicase domain with conserved motifs that are essential for ATP binding and hydrolysis, RNA interaction, and unwinding activities. Large families of DEAD/DExH-box proteins have been described in different organisms, and their role in all molecular processes involving RNA, from transcriptional regulation to mRNA decay, have been described. This review aims to summarize the current knowledge about DEAD/DExH-box proteins in selected protozoan and nematode parasites of medical importance worldwide, such as Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Giardia lamblia, Entamoeba histolytica, and Brugia malayi. We discuss the functional characterization of several proteins in an attempt to understand better the molecular mechanisms involving RNA in these pathogens. The current data also highlight that DEAD/DExH-box RNA helicases might represent feasible drug targets due to their vital role in parasite growth and development.

  8. DEAD/DExH-Box RNA Helicases in Selected Human Parasites

    PubMed Central

    Marchat, Laurence A.; Arzola-Rodríguez, Silvia I.; Hernandez-de la Cruz, Olga; Lopez-Rosas, Itzel; Lopez-Camarillo, Cesar

    2015-01-01

    DEAD/DExH-box RNA helicases catalyze the folding and remodeling of RNA molecules in prokaryotic and eukaryotic cells, as well as in many viruses. They are characterized by the presence of the helicase domain with conserved motifs that are essential for ATP binding and hydrolysis, RNA interaction, and unwinding activities. Large families of DEAD/DExH-box proteins have been described in different organisms, and their role in all molecular processes involving RNA, from transcriptional regulation to mRNA decay, have been described. This review aims to summarize the current knowledge about DEAD/DExH-box proteins in selected protozoan and nematode parasites of medical importance worldwide, such as Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Giardia lamblia, Entamoeba histolytica, and Brugia malayi. We discuss the functional characterization of several proteins in an attempt to understand better the molecular mechanisms involving RNA in these pathogens. The current data also highlight that DEAD/DExH-box RNA helicases might represent feasible drug targets due to their vital role in parasite growth and development. PMID:26537038

  9. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  10. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato.

    PubMed

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants. PMID:26241658

  11. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato

    PubMed Central

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants. PMID:26241658

  12. The helC gene encodes a putative DEAD-box RNA helicase required for development in Dictyostelium discoideum.

    PubMed

    Machesky, L M; Insall, R H; Kay, R R

    1998-05-01

    DEAD-box RNA helicases, defined by the sequence Asp-Glu-Ala-Asp (DEAD, in single-letter amino-acid code), regulate RNA unwinding and secondary structure in an ATP-dependent manner in vitro [1] and control mRNA stability and protein translation. Both yeast and mammals have large families of DEAD-box proteins, many of unknown function. We have disrupted a Dictyostelium discoideum gene, helC, which encodes helicase C, a member of the DEAD-box family of RNA helicases that shows strong homology to the product of the essential Saccharomyces cerevisiae gene dbp5 [2] and to related helicases in mouse and Schizosaccharomyces pombe. The HelC protein also shows weaker homology to the translation initiation factor elF-4a. Other DEAD-box-containing proteins, which are less closely related to HelC, have been implicated in developmental roles in Drosophila [3] and Xenopus laevis; one example is the Xenopus Vasa-like protein (XVLP) [4-6]. In Drosophila and Xenopus, Vasa and XVLP, respectively, are required for the establishment of tissue polarity during development. In yeast, DEAD-box helicases such as Prp8 [7] are components of the spliceosome and connect pre-mRNA splicing with the cell cycle. Disruption of the helC gene in D. discoideum led to developmental asynchrony, failure to differentiate and aberrant morphogenesis. We postulate that one reason for the existence of large families of homologous DEAD-box proteins in yeast, mammals and Dictyostelium could be that some DEAD-box proteins have developmentally specific roles regulating protein translation or mRNA stability. PMID:9601648

  13. The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases.

    PubMed Central

    Sowden, J; Putt, W; Morrison, K; Beddington, R; Edwards, Y

    1995-01-01

    DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8948440

  14. NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47

    PubMed Central

    Sekiguchi, Takeshi; Hayano, Toshiya; Yanagida, Mitsuaki; Takahashi, Nobuhiro; Nishimoto, Takeharu

    2006-01-01

    Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus. PMID:16963496

  15. Structure-guided mutational analysis of a yeast DEAD-box protein involved in mitochondrial RNA splicing.

    PubMed

    Bifano, Abby L; Turk, Edward M; Caprara, Mark G

    2010-05-01

    DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context

  16. Structure-Guided Mutational Analysis of a Yeast DEAD-box Protein Involved in Mitochondrial RNA Splicing

    PubMed Central

    Bifano, Abby L.; Turk, Edward M.; Caprara, Mark G.

    2010-01-01

    Summary DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, bind nucleic acid, and promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probe the importance of several highly conserved residues in the yeast DEAD-box protein, Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using a MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observe that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context

  17. Nucleolar DEAD-Box RNA Helicase TOGR1 Regulates Thermotolerant Growth as a Pre-rRNA Chaperone in Rice.

    PubMed

    Wang, Dong; Qin, Baoxiang; Li, Xiang; Tang, Ding; Zhang, Yu'e; Cheng, Zhukuan; Xue, Yongbiao

    2016-02-01

    Plants have evolved a considerable number of intrinsic tolerance strategies to acclimate to ambient temperature increase. However, their molecular mechanisms remain largely obscure. Here we report a DEAD-box RNA helicase, TOGR1 (Thermotolerant Growth Required1), prerequisite for rice growth themotolerance. Regulated by both temperature and the circadian clock, its expression is tightly coupled to daily temperature fluctuations and its helicase activities directly promoted by temperature increase. Located in the nucleolus and associated with the small subunit (SSU) pre-rRNA processome, TOGR1 maintains a normal rRNA homeostasis at high temperature. Natural variation in its transcript level is positively correlated with plant height and its overexpression significantly improves rice growth under hot conditions. Our findings reveal a novel molecular mechanism of RNA helicase as a key chaperone for rRNA homeostasis required for rice thermotolerant growth and provide a potential strategy to breed heat-tolerant crops by modulating the expression of TOGR1 and its orthologs. PMID:26848586

  18. Nucleolar DEAD-Box RNA Helicase TOGR1 Regulates Thermotolerant Growth as a Pre-rRNA Chaperone in Rice.

    PubMed

    Wang, Dong; Qin, Baoxiang; Li, Xiang; Tang, Ding; Zhang, Yu'e; Cheng, Zhukuan; Xue, Yongbiao

    2016-02-01

    Plants have evolved a considerable number of intrinsic tolerance strategies to acclimate to ambient temperature increase. However, their molecular mechanisms remain largely obscure. Here we report a DEAD-box RNA helicase, TOGR1 (Thermotolerant Growth Required1), prerequisite for rice growth themotolerance. Regulated by both temperature and the circadian clock, its expression is tightly coupled to daily temperature fluctuations and its helicase activities directly promoted by temperature increase. Located in the nucleolus and associated with the small subunit (SSU) pre-rRNA processome, TOGR1 maintains a normal rRNA homeostasis at high temperature. Natural variation in its transcript level is positively correlated with plant height and its overexpression significantly improves rice growth under hot conditions. Our findings reveal a novel molecular mechanism of RNA helicase as a key chaperone for rRNA homeostasis required for rice thermotolerant growth and provide a potential strategy to breed heat-tolerant crops by modulating the expression of TOGR1 and its orthologs.

  19. Nucleolar DEAD-Box RNA Helicase TOGR1 Regulates Thermotolerant Growth as a Pre-rRNA Chaperone in Rice

    PubMed Central

    Tang, Ding; Zhang, Yu’e; Cheng, Zhukuan; Xue, Yongbiao

    2016-01-01

    Plants have evolved a considerable number of intrinsic tolerance strategies to acclimate to ambient temperature increase. However, their molecular mechanisms remain largely obscure. Here we report a DEAD-box RNA helicase, TOGR1 (Thermotolerant Growth Required1), prerequisite for rice growth themotolerance. Regulated by both temperature and the circadian clock, its expression is tightly coupled to daily temperature fluctuations and its helicase activities directly promoted by temperature increase. Located in the nucleolus and associated with the small subunit (SSU) pre-rRNA processome, TOGR1 maintains a normal rRNA homeostasis at high temperature. Natural variation in its transcript level is positively correlated with plant height and its overexpression significantly improves rice growth under hot conditions. Our findings reveal a novel molecular mechanism of RNA helicase as a key chaperone for rRNA homeostasis required for rice thermotolerant growth and provide a potential strategy to breed heat-tolerant crops by modulating the expression of TOGR1 and its orthologs. PMID:26848586

  20. Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster.

    PubMed

    Surabhi, Satya; Tripathi, Bipin K; Maurya, Bhawana; Bhaskar, Pradeep K; Mukherjee, Ashim; Mutsuddi, Mousumi

    2015-11-01

    Notch signaling is an evolutionary conserved process that influences cell fate determination, cell proliferation, and cell death in a context-dependent manner. Notch signaling is fine-tuned at multiple levels and misregulation of Notch has been implicated in a variety of human diseases. We have characterized maheshvara (mahe), a novel gene in Drosophila melanogaster that encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. A dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe in a wide range of tissues during development results in a variety of defects, many of which resemble a typical Notch loss-of-function phenotype. We illustrate that ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch-mediated proliferation of eye tissue. Further, we illustrate that mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Coexpression of Deltex and Mahe at the dorso-ventral boundary results in a wing-nicking phenotype and a more pronounced loss of Notch target Cut. Taken together we report identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signaling.

  1. Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

    PubMed Central

    Vakulskas, Christopher A.; Pannuri, Archana; Cortés-Selva, Diana; Zere, Tesfalem R.; Ahmer, Brian M.; Babitzke, Paul; Romeo, Tony

    2014-01-01

    Summary In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two noncoding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance basepairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function. PMID:24708042

  2. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature

    PubMed Central

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold. PMID:27116354

  3. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature.

    PubMed

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.

  4. DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

    PubMed

    Soltanieh, Sahar; Osheim, Yvonne N; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L; Dragon, François

    2015-03-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

  5. A human gene (DDX10) encoding a putative DEAD-box RNA helicase at 11q22-q23

    SciTech Connect

    Savitsky, K.; Ziv, Y.; Bar-Shira, A.

    1996-04-15

    A human gene encoding a putative RNA helicase, designated DDX10, was identified 400 kb telomeric to the ataxia-telangiectasia gene at chromosome 11q22-q23. The predicted amino acid sequence shows very high similarity to a subgroup of DEAD-box RNA helicases involved in ribosome biogenesis. This novel gene encodes a 3.2-kb transcript in a variety of human tissues. A processed pseudogene of DDX10 was detected at chromosome 9q21-q22. We observed a rare trinucleotide repeat length polymorphism within the coding sequence of DDX10. 39 refs., 5 figs.

  6. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

    PubMed Central

    Pause, A; Sonenberg, N

    1992-01-01

    eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity. Images PMID:1378397

  7. Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

    PubMed

    Liu, Chenlin; Huang, Xiaohang

    2015-09-01

    DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation.

  8. Yeast DEAD box protein Mss116p is a transcription elongation factor that modulates the activity of mitochondrial RNA polymerase.

    PubMed

    Markov, Dmitriy A; Wojtas, Ireneusz D; Tessitore, Kassandra; Henderson, Simmone; McAllister, William T

    2014-07-01

    DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. PMID:24732805

  9. DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition

    PubMed Central

    Samatanga, Brighton; Klostermeier, Dagmar

    2014-01-01

    DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes. PMID:25123660

  10. The requirement of the DEAD-box protein DDX24 for the packaging of human immunodeficiency virus type 1 RNA

    SciTech Connect

    Ma Jing; Rong Liwei; Zhou Yongdong; Roy, Bibhuti Bushan; Lu, Jennifer; Abrahamyan, Levon; Mouland, Andrew J.; Pan Qinghua; Chen Liang

    2008-05-25

    RNA helicases play important roles in RNA metabolism. Human immunodeficiency virus type 1 (HIV-1) does not carry its own RNA helicase, the virus thus needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. In this study, we report that knockdown of a DEAD-box protein named DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.

  11. When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

    PubMed

    Rudolph, Markus G; Klostermeier, Dagmar

    2015-08-01

    DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

  12. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA.

    PubMed

    Nishimura, Kenji; Ashida, Hiroki; Ogawa, Taro; Yokota, Akiho

    2010-09-01

    In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

  13. The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly

    PubMed Central

    Tu, Ya-Ting; Barrientos, Antoni

    2015-01-01

    SUMMARY Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process and factors involved remain largely unknown. Here, we have identified the DEAD box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability, 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that mainly takes place near the mitochondrial nucleoids, in the compartment defined by the RNA granules. PMID:25683708

  14. The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality.

    PubMed

    Radhakrishnan, Aditya; Chen, Ying-Hsin; Martin, Sophie; Alhusaini, Najwa; Green, Rachel; Coller, Jeff

    2016-09-22

    A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay. PMID:27641505

  15. Coupling between the DEAD-box RNA helicases Ded1p and eIF4A

    PubMed Central

    Gao, Zhaofeng; Putnam, Andrea A; Bowers, Heath A; Guenther, Ulf-Peter; Ye, Xuan; Kindsfather, Audrey; Hilliker, Angela K; Jankowsky, Eckhard

    2016-01-01

    Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. Here we show that S. cerevisiae eIF4A and Ded1p directly interact with each other and simultaneously with the scaffolding protein eIF4G. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as a modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E. DOI: http://dx.doi.org/10.7554/eLife.16408.001 PMID:27494274

  16. A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold-Responsive Gene Regulation, and Cold Tolerance in Arabidopsis[C][W

    PubMed Central

    Guan, Qingmei; Wu, Jianmin; Zhang, Yanyan; Jiang, Changhua; Liu, Renyi; Chai, Chenglin; Zhu, Jianhua

    2013-01-01

    Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis thaliana mutants for deregulated expression of a firefly luciferase reporter gene under the control of the C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC). A regulator of CBF gene expression1 (rcf1-1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Ala-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES4 [LOS4]) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (PSEUDO-RESPONSE REGULATOR5 [PRR5], SHAGGY-LIKE SERINE/THREONINE KINASE12 [SK12], MYB FAMILY TRANSCRIPTION FACTOR CIRCADIAN1 [CIR1], and SPFH/PHB DOMAIN-CONTAINING MEMBRANE-ASSOCIATED PROTEIN [SPFH]) that are mis-spliced in rcf1-1 revealed that these genes are cold-inducible positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants. PMID:23371945

  17. The DEAD Box RNA helicase VBH-1 is a new player in the stress response in C. elegans.

    PubMed

    Paz-Gómez, Daniel; Villanueva-Chimal, Emmanuel; Navarro, Rosa E

    2014-01-01

    For several years, DEAD box RNA helicase Vasa (DDX4) has been used as a bona fide germline marker in different organisms. C. elegans VBH-1 is a close homolog of the Vasa protein, which plays an important role in gametogenesis, germ cell survival and embryonic development. Here, we show that VBH-1 protects nematodes from heat shock and oxidative stress. Using the germline-defective mutant glp-4(bn2) we found that a potential somatic expression of vbh-1 might be important for stress survival. We also show that the VBH-1 paralog LAF-1 is important for stress survival, although this protein is not redundant with its counterpart. Furthermore, we observed that the mRNAs of the heat shock proteins hsp-1 and sip-1 are downregulated when vbh-1 or laf-1 are silenced. Previously, we reported that in C. elegans, VBH-1 was primarily expressed in P granules of germ cells and in the cytoplasm of all blastomeres. Here we show that during stress, VBH-1 co-localizes with CGH-1 in large aggregates in the gonad core and oocytes; however, VBH-1 aggregates do not overlap with CGH-1 foci in early embryos under the same conditions. These data demonstrate that, in addition to the previously described role for this protein in the germline, VBH-1 plays an important role during the stress response in C. elegans through the potential direct or indirect regulation of stress response mRNAs.

  18. SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo

    PubMed Central

    Trubetskoy, Dmitrii; Proux, Florence; Allemand, Frédéric; Dreyfus, Marc; Iost, Isabelle

    2009-01-01

    DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5′-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly. PMID:19734346

  19. SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo.

    PubMed

    Trubetskoy, Dmitrii; Proux, Florence; Allemand, Frédéric; Dreyfus, Marc; Iost, Isabelle

    2009-10-01

    DEAD-box proteins play specific roles in remodeling RNA or ribonucleoprotein complexes. Yet, in vitro, they generally behave as nonspecific RNA-dependent ATPases, raising the question of what determines their specificity in vivo. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit. Moreover, when overexpressed, it compensates for a mutation in L24, the ribosomal protein (r-protein) thought to initiate assembly. Here, using the tandem affinity purification (TAP) procedure, we show that SrmB forms a complex with r-proteins L4, L24 and a region near the 5'-end of 23S rRNA that binds these proteins. In vitro reconstitution experiments show that the stability of this complex reflects cooperative interactions of SrmB with L4, L24 and rRNA. These observations are consistent with an early role of SrmB in assembly and explain the genetic link between SrmB and L24. Besides its catalytic core, SrmB possesses a nonconserved C-terminal extension that, we show, is not essential for SrmB function and specificity. In this regard, SrmB differs from DbpA, another DEAD-box protein involved in ribosome assembly.

  20. A novel function for the DEAD-box RNA helicase DDX-23 in primary microRNA processing in Caenorhabditis elegans.

    PubMed

    Chu, Yu-De; Chen, Hsin-Kai; Huang, Tao; Chan, Shih-Peng

    2016-01-15

    Primary microRNAs (pri-miRNAs) are cleaved by the nuclear RNase III Drosha to produce hairpin-shaped precursor miRNAs (pre-miRNAs). In humans, this process is known to be facilitated by the DEAD-box helicases p68 (DDX5) and p72 (DDX17). In this study, we performed a candidate-based RNAi screen in C. elegans to identify DEAD/H-box proteins involved in miRNA biogenesis. In a let-7(mg279) sensitized genetic background, knockdown of a homolog of yeast splicing factor Prp28p, DDX-23, or a homolog of human helicases p68 and p72, DDX-17, enhanced let-7 loss-of-function phenotypes, suggesting that these helicases play a role in let-7 processing and/or function. In both ddx-23(RNAi) and ddx-17(RNAi), levels of mature let-7 were decreased while pri-let-7 was found to accumulate, indicating that the helicases likely act at the level of pri-let-7 processing. DDX-23 and DDX-17 were also required for the biogenesis of other known heterochronic miRNAs, including lin-4 and the let-7 family members miR-48, miR-84 and miR-241. Their function was not confined to the heterochronic pathway, however, since they were both necessary for down-regulation of cog-1 by the spatial patterning miRNA, lsy-6. Here, we present a novel function for C. elegans DDX-23 in pri-miRNA processing, and also suggest a conserved role for DDX-17 in this process.

  1. Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

    PubMed Central

    Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

    2010-01-01

    CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

  2. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    SciTech Connect

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.

  3. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    DOE PAGES

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to themore » adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.« less

  4. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    SciTech Connect

    Kellner, Julian N.; Meinhart, Anton

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  5. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein.

    PubMed

    Kellner, Julian N; Meinhart, Anton

    2015-09-01

    The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein-protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein-protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.

  6. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    PubMed Central

    Kellner, Julian N.; Meinhart, Anton

    2015-01-01

    The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor. PMID:26323305

  7. Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue.

    PubMed

    Kaushik, Ramakant; Singh, Karn Pratap; Bahuguna, Vivek; Rameshbabu, K; Singh, Manoj Kumar; Manik, Radhey Shyam; Palta, Prabhat; Singla, Suresh Kumar; Chauhan, Manmohan Singh

    2015-11-01

    VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3'-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P < 0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.

  8. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    SciTech Connect

    Kellner, Markus; Rohrmoser, Michaela; Forné, Ignasi; Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  9. The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription

    PubMed Central

    Gao, Yu; Song, Feifei; Guo, Liang; Ma, Li; Sun, Guihong; Liu, Dan; Guo, Deyin

    2016-01-01

    RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65. PMID:27736973

  10. Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

    PubMed Central

    Alessi, Amelia F.; Khivansara, Vishal; Han, Ting; Freeberg, Mallory A.; Moresco, James J.; Tu, Patricia G.; Montoye, Eric; Yates, John R.; Karp, Xantha; Kim, John K.

    2015-01-01

    MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway. PMID:26669440

  11. Characterization of the kinetics of RNA annealing and strand displacement activities of the E. coli DEAD-box helicase CsdA.

    PubMed

    Stampfl, Sabine; Doetsch, Martina; Beich-Frandsen, Mads; Schroeder, Renée

    2013-01-01

    CsdA is one of five E. coli DEAD-box helicases and as a cold-shock protein assists RNA structural remodeling at low temperatures. The helicase has been shown to catalyze duplex unwinding in an ATP-dependent way and accelerate annealing of complementary RNAs, but detailed kinetic analyses are missing. Therefore, we performed kinetic measurements using a coupled annealing and strand displacement assay with high temporal resolution to analyze how CsdA balances the two converse activities. We furthermore tested the hypothesis that the unwinding activity of DEAD-box helicases is largely determined by the substrate's thermodynamic stability using full-length CsdA and a set of RNAs with constant length, but increasing GC content. The rate constants for strand displacement did indeed decrease with increasing duplex stability, with a calculated free energy between -31.3 and -40 kcal/mol being the limit for helix unwinding. Thus, our data generally support the above hypothesis, showing that for CsdA substrate thermal stability is an important rate limiting factor.

  12. P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).

    PubMed

    Wong, Emily V; Cao, Wenxiang; Vörös, Judit; Merchant, Monique; Modis, Yorgo; Hackney, David D; Montpetit, Ben; De La Cruz, Enrique M

    2016-01-29

    mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors. PMID:26730886

  13. P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).

    PubMed

    Wong, Emily V; Cao, Wenxiang; Vörös, Judit; Merchant, Monique; Modis, Yorgo; Hackney, David D; Montpetit, Ben; De La Cruz, Enrique M

    2016-01-29

    mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.

  14. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2009-09-02

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/{Delta}598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions ({le}1 mg ml{sup -1}), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of {approx}10 mg ml{sup -1}. Initial crystals were obtained by the microbatch method in the presence of a U{sub 10} RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/{Delta}598-664 in complex with AMP-PNP and U{sub 10} belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 {angstrom}, and diffracted X-rays to beyond 1.9 {angstrom} resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.

  15. Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis.

    PubMed

    Asakura, Yukari; Galarneau, Erin; Watkins, Kenneth P; Barkan, Alice; van Wijk, Klaas J

    2012-07-01

    Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

  16. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    SciTech Connect

    Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer DP97 interacts with nuclear receptor CAR. Black-Right-Pointing-Pointer DP97 enhances CAR-mediated transcriptional activation. Black-Right-Pointing-Pointer DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1{alpha}. Black-Right-Pointing-Pointer DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1{alpha}, therefore it might act as mediator between hCAR and appropriate co-activators.

  17. The plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.

    PubMed

    Kanai, Masatake; Hayashi, Makoto; Kondo, Maki; Nishimura, Mikio

    2013-09-01

    Plants accumulate large amounts of storage products in seeds to provide an energy reserve and to supply nutrients for germination and post-germinative growth. Arabidopsis thaliana belongs to the Brassica family, and oil is the main storage product in Arabidopsis seeds. To elucidate the regulatory mechanisms of oil biosynthesis in seeds, we screened for high density seeds (heavy seed) that have a low oil content. HS3 (heavy seed 3) encodes the DEAD-box RNA helicase 22 that is localized to plastids. The triacylglycerol (TAG) content of hs3-1 seeds was 10% lower than that of wild-type (WT) seeds, while the protein content was unchanged. The hs3-1 plants displayed a pale-green phenotype in developing seeds and seedlings, but not in adult leaves. The HS3 expression level was high in developing seeds and seedlings, but was low in stems, rosette leaves and flowers. The plastid gene expression profile of WT developing seeds and seedlings differed from that of hs3-1 developing seeds and seedlings. The expression of several genes was reduced in developing hs3-1 seeds, including accD, a gene that encodes the β subunit of carboxyltransferase, which is one component of acetyl-CoA carboxylase in plastids. In contrast, no differences were observed between the expression profiles of WT and hs3-1 rosette leaves. These results show that HS3 is essential for proper mRNA accumulation of plastid genes during seed development and seedling growth, and suggest that HS3 ensures seed oil biosynthesis by maintaining plastid mRNA levels.

  18. SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

    PubMed Central

    Solana, Jordi; Romero, Rafael

    2009-01-01

    Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids. PMID:19159016

  19. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

    PubMed Central

    Çelik, Haydar; Sajwan, Kamal P.; Selvanathan, Saravana P.; Marsh, Benjamin J.; Pai, Amrita V.; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z.; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A.

    2015-01-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. PMID:26149384

  20. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    PubMed

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  1. AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose

    PubMed Central

    Hsu, Yi-Feng; Chen, Yun-Chu; Hsiao, Yu-Chun; Wang, Bing-Jyun; Lin, Shih-Yun; Cheng, Wan-Hsing; Jauh, Guang-Yuh; Harada, John J; Wang, Co-Shine

    2014-01-01

    The Arabidopsis thalianaT-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth. PMID:24176057

  2. AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

    PubMed

    Hsu, Yi-Feng; Chen, Yun-Chu; Hsiao, Yu-Chun; Wang, Bing-Jyun; Lin, Shih-Yun; Cheng, Wan-Hsing; Jauh, Guang-Yuh; Harada, John J; Wang, Co-Shine

    2014-01-01

    The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

  3. AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

    PubMed

    Hsu, Yi-Feng; Chen, Yun-Chu; Hsiao, Yu-Chun; Wang, Bing-Jyun; Lin, Shih-Yun; Cheng, Wan-Hsing; Jauh, Guang-Yuh; Harada, John J; Wang, Co-Shine

    2014-01-01

    The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth. PMID:24176057

  4. Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

    PubMed

    Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

    2010-03-01

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.

  5. Amplification of a DEAD box protein gene in retinoblastoma cell lines.

    PubMed Central

    Godbout, R; Squire, J

    1993-01-01

    DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp, are putative RNA helicases implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. Here, we report that the mRNA encoding a DEAD box protein, designated HuDBP-RB, is present at elevated levels in two of six retinoblastoma (RB) cell lines tested and is preferentially expressed in fetal tissues of neuroectodermal origin. It is not possible to classify HuDBP-RB as a member of any of the DEAD box protein subgroups identified to date since the regions of amino acid similarity between HuDBP-RB and other DEAD box proteins are restricted to the conserved motifs found in all members of this family. The HuDBP-RB gene, which has been mapped to chromosome band 2p24, is amplified in the RB cell lines that overexpress HuDBP-RB RNA. Furthermore, the MYCN gene is also present in multiple copies in these two cell lines, suggesting coamplification of the two genes. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7689221

  6. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  7. Roles of Four Putative DEAD-Box RNA Helicase Genes in Growth of Listeria monocytogenes EGD-e under Heat, pH, Osmotic, Ethanol, and Oxidative Stress Conditions

    PubMed Central

    Lindström, Miia; Johansson, Per; Björkroth, Johanna; Korkeala, Hannu

    2012-01-01

    To examine the role of the four putative DEAD-box RNA helicase genes of Listeria monocytogenes EGD-e in stress tolerance, the growth of the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 deletion mutant strains at 42.5°C, at pH 5.6 or pH 9.4, in 6% NaCl, in 3.5% ethanol, and in 5 mM H2O2 was studied. Restricted growth of the Δlmo0866 deletion mutant strain in 3.5% ethanol suggests that Lmo0866 contributes to ethanol stress tolerance of L. monocytogenes EGD-e. The Δlmo1450 mutant strain showed negligible growth at 42.5°C, at pH 9.4, and in 5 mM H2O2 and a lower maximum growth temperature than the wild-type EGD-e, suggesting that Lmo1450 is involved in the tolerance of L. monocytogenes EGD-e to heat, alkali, and oxidative stresses. The altered stress tolerance of the Δlmo0866 and Δlmo1450 deletion mutant strains did not correlate with changes in relative expression levels of lmo0866 and lmo1450 genes under corresponding stresses, suggesting that Lmo0866- and Lmo1450-dependent tolerance to heat, alkali, ethanol, or oxidative stress is not regulated at the transcriptional level. Growth of the Δlmo1246 and Δlmo1722 deletion mutant strains did not differ from that of the wild-type EGD-e under any of the conditions tested, suggesting that Lmo1246 and Lmo1722 have no roles in the growth of L. monocytogenes EGD-e under heat, pH, osmotic, ethanol, or oxidative stress. This study shows that the putative DEAD-box RNA helicase genes lmo0866 and lmo1450 play important roles in tolerance of L. monocytogenes EGD-e to ethanol, heat, alkali, and oxidative stresses. PMID:22820328

  8. The DEAD-Box RNA Helicase DDX3 Associates with Export Messenger Ribonucleoproteins as well asTip-associated Protein and Participates in Translational Control

    PubMed Central

    Lai, Ming-Chih; Lee, Yan-Hwa Wu

    2008-01-01

    Nuclear export of mRNA is tightly linked to transcription, nuclear mRNA processing, and subsequent maturation in the cytoplasm. Tip-associated protein (TAP) is the major nuclear mRNA export receptor, and it acts coordinately with various factors involved in mRNA expression. We screened for protein factors that associate with TAP and identified several candidates, including RNA helicase DDX3. We demonstrate that DDX3 directly interacts with TAP and that its association with TAP as well as mRNA ribonucleoprotein complexes may occur in the nucleus. Depletion of TAP resulted in nuclear accumulation of DDX3, suggesting that DDX3 is, at least in part, exported along with messenger ribonucleoproteins to the cytoplasm via the TAP-mediated pathway. Moreover, the observation that DDX3 localizes transiently in cytoplasmic stress granules under cell stress conditions suggests a role for DDX3 in translational control. Indeed, DDX3 associates with translation initiation complexes. However, DDX3 is probably not critical for general mRNA translation but may instead promote efficient translation of mRNAs containing a long or structured 5′ untranslated region. Given that the DDX3 RNA helicase activity is essential for its involvement in translation, we suggest that DDX3 facilitates translation by resolving secondary structures of the 5′-untranslated region in mRNAs during ribosome scanning. PMID:18596238

  9. Evolution of the DEAD box helicase family in chicken: chickens have no DHX9 ortholog.

    PubMed

    Sato, Haruko; Oshiumi, Hiroyuki; Takaki, Hiromi; Hikono, Hirokazu; Seya, Tsukasa

    2015-10-01

    Viral RNA represents a pattern molecule that can be recognized by RNA sensors in innate immunity. Humans and mice possess cytoplasmic DNA/RNA sensors for detecting viral replication. There are a number of DEAD (Asp-Glu-Ala-Asp; DExD/H) box-type helicases in mammals, among which retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA50) are indispensable for RNA sensing; however, they are functionally supported by a number of sensors that directly bind viral RNA or replicative RNA intermediates to convey signals to RIG-I and MDA5. Some DEAD box helicase members recognize DNA irrespective of the origin. These sensors transmit IFN-inducing signals through adaptors, including mitochondrial antiviral signaling. Viral double-stranded RNAs are reportedly sensed by the helicases DDX1, DDX21, DHX36, DHX9, DDX3, DDX41, LGP2 and DDX60, in addition to RIG-I and MDA5, and induce type I IFNs, thereby blocking viral replication. Humans and mice have all nucleic acid sensors listed here. In the RNA sensing system in chicken, it was found in the present study that most DEAD box helicases are conserved; however, DHX9 is genetically deficient in addition to reported RIG-I. Based on the current genome databases, similar DHX9 deficiency was observed in ducks and several other bird species. Because chicken, but not duck, was found to be deficient in RIG-I, the RNA-sensing system of chicken lacks RIG-I and DHX9 and is thus more fragile than that of duck or mammal. DHX9 may generally compensate for the function of RIG-I and deficiency of DHX9 possibly participates in exacerbations of viral infection such as influenza in chickens.

  10. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

    PubMed

    Grandori, C; Mac, J; Siëbelt, F; Ayer, D E; Eisenman, R N

    1996-08-15

    The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism.

  11. The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.

    PubMed

    De Silva, Dasmanthie; Fontanesi, Flavia; Barrientos, Antoni

    2013-11-01

    Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions.

  12. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

    SciTech Connect

    Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2012-03-15

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.

  13. Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3' UTR structures.

    PubMed

    Ward, Alex Michael; Bidet, Katell; Yinglin, Ang; Ler, Siok Ghee; Hogue, Kelly; Blackstock, Walter; Gunaratne, Jayantha; Garcia-Blanco, Mariano A

    2011-01-01

    Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines.  The DENV-2 positive-strand RNA genome contains 5' and 3' untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins.  In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5' and 3' UTRs as "bait", combined with quantitative mass spectrometry.  We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs.  Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex.  Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3' UTR, possibly by formation of a pseudoknot structure.  Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3' UTR.  These results suggest that the DENV-2 3' UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3' UTR is required for DENV replication.

  14. Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI.

    PubMed

    Patel, Krishnaben; Shah, Grishma K; Kommaraju, Sai Shilpa; Low, Woon-Kai

    2014-02-01

    The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AI-binding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A. PMID:24471916

  15. Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI.

    PubMed

    Patel, Krishnaben; Shah, Grishma K; Kommaraju, Sai Shilpa; Low, Woon-Kai

    2014-02-01

    The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AI-binding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A.

  16. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    SciTech Connect

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  17. DEAD-box proteins, like Leishmania eIF4A, modulate interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha production by human monocytes.

    PubMed

    Barhoumi, M; Meddeb-Garnaoui, A; Tanner, N K; Banroques, J; Kaabi, B; Guizani, I

    2013-01-01

    Previously we showed that His-tagged, recombinant, Leishmania infantum eukaryotic initiation factor (LeIF) was both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described for other members of the DEAD-box helicase family. In addition, we showed that LeIF induces the production of IL-12, IL-10, and TNF-α by human monocytes. This study aims to characterize the cytokine-inducing activity in human monocytes of several proteins belonging to the DEAD-box family from mammals and yeast. All tested proteins contained the 11 conserved motifs (Q, I, Ia, GG Ib, II, III, IV, QxxR, V and VI) characteristic of DEAD-box proteins, but they have different biological functions and different percentages of identities with LeIF. We show that these mammalian or yeast recombinant proteins also are able to induce IL-12, IL-10 and TNF-α secretion by monocytes of healthy human subjects. This cytokine-inducing activity is proteinase K sensitive and polymyxin B resistant. Our results show that the induction of cytokines in human monocytes is not unique to the protein LeIF of Leishmania, and it suggests that the activity of certain DEAD-box proteins can be exploited as adjuvant and/or to direct immune responses towards a Th1 profile in vaccination or immunotherapy protocols. PMID:23363368

  18. Structural and Functional Analysis of the Interaction Between the Nucleoporin Nup214 and the DEAD-box Helicase Ddx19

    SciTech Connect

    Napetschnig, J.; Kassube, S; Debler, E; Wong, R; Blobel, G; Hoelz, A

    2009-01-01

    Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 {angstrom} resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.

  19. Structural and functional analysis of the interaction between the nucleoporin Nup214 and the DEAD-box helicase Ddx19

    PubMed Central

    Napetschnig, Johanna; Kassube, Susanne A.; Debler, Erik W.; Wong, Richard W.; Blobel, Günter; Hoelz, André

    2009-01-01

    Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 Å resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export. PMID:19208808

  20. Cloning, characterization, and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp ( Fenneropenaeus chinensis)

    NASA Astrophysics Data System (ADS)

    Zhou, Qianru; Shao, Mingyu; Qin, Zhenkui; Kyoung, Ho Kang; Zhang, Zhifeng

    2010-01-01

    RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-vasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.

  1. Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor.

    PubMed

    Iwasaki, Shintaro; Floor, Stephen N; Ingolia, Nicholas T

    2016-06-23

    Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions. PMID:27309803

  2. Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor.

    PubMed

    Iwasaki, Shintaro; Floor, Stephen N; Ingolia, Nicholas T

    2016-06-15

    Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.

  3. Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

    2013-09-01

    DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

  4. DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

    SciTech Connect

    Kim, Ki Hyung; Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun; Moon, Soo Hyun; Suh, Dong Soo; Yoon, Man Soo; Park, Eun-Sil; Jeong, Namkung; Eo, Wan-Kyu; Kim, Heung Yeol; Cha, Hee-Jae

    2014-05-02

    Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

  5. The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

    PubMed Central

    Senissar, Meriem; Saux, Agnès Le; Belgareh-Touzé, Naïma; Adam, Céline; Banroques, Josette; Tanner, N. Kyle

    2014-01-01

    The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs. PMID:25013175

  6. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

    PubMed Central

    Khoshnevis, Sohail; Askenasy, Isabel; Dattolo, Maria D.; Young-Erdos, Crystal L.; Stroupe, M. Elizabeth; Karbstein, Katrin

    2016-01-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  7. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    PubMed

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  8. Identification of human miRNA precursors that resemble box C/D snoRNAs.

    PubMed

    Ono, Motoharu; Scott, Michelle S; Yamada, Kayo; Avolio, Fabio; Barton, Geoffrey J; Lamond, Angus I

    2011-05-01

    There are two main classes of small nucleolar RNAs (snoRNAs): the box C/D snoRNAs and the box H/ACA snoRNAs that function as guide RNAs to direct sequence-specific modification of rRNA precursors and other nucleolar RNA targets. A previous computational and biochemical analysis revealed a possible evolutionary relationship between miRNA precursors and some box H/ACA snoRNAs. Here, we investigate a similar evolutionary relationship between a subset of miRNA precursors and box C/D snoRNAs. Computational analyses identified 84 intronic miRNAs that are encoded within either box C/D snoRNAs, or in precursors showing similarity to box C/D snoRNAs. Predictions of the folded structures of these box C/D snoRNA-like miRNA precursors resemble the structures of known box C/D snoRNAs, with the boxes C and D often in close proximity in the folded molecule. All five box C/D snoRNA-like miRNA precursors tested (miR-27b, miR-16-1, mir-28, miR-31 and let-7g) bind to fibrillarin, a specific protein component of functional box C/D snoRNP complexes. The data suggest that a subset of small regulatory RNAs may have evolved from box C/D snoRNAs.

  9. Human miRNA Precursors with Box H/ACA snoRNA Features

    PubMed Central

    Scott, Michelle S.; Avolio, Fabio; Ono, Motoharu; Lamond, Angus I.; Barton, Geoffrey J.

    2009-01-01

    MicroRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) are two classes of small non-coding regulatory RNAs, which have been much investigated in recent years. While their respective functions in the cell are distinct, they share interesting genomic similarities, and recent sequencing projects have identified processed forms of snoRNAs that resemble miRNAs. Here, we investigate a possible evolutionary relationship between miRNAs and box H/ACA snoRNAs. A comparison of the genomic locations of reported miRNAs and snoRNAs reveals an overlap of specific members of these classes. To test the hypothesis that some miRNAs might have evolved from snoRNA encoding genomic regions, reported miRNA-encoding regions were scanned for the presence of box H/ACA snoRNA features. Twenty miRNA precursors show significant similarity to H/ACA snoRNAs as predicted by snoGPS. These include molecules predicted to target known ribosomal RNA pseudouridylation sites in vivo for which no guide snoRNA has yet been reported. The predicted folded structures of these twenty H/ACA snoRNA-like miRNA precursors reveal molecules which resemble the structures of known box H/ACA snoRNAs. The genomic regions surrounding these predicted snoRNA-like miRNAs are often similar to regions around snoRNA retroposons, including the presence of transposable elements, target site duplications and poly (A) tails. We further show that the precursors of five H/ACA snoRNA-like miRNAs (miR-151, miR-605, mir-664, miR-215 and miR-140) bind to dyskerin, a specific protein component of functional box H/ACA small nucleolar ribonucleoprotein complexes suggesting that these molecules have retained some H/ACA snoRNA functionality. The detection of small RNA molecules that share features of miRNAs and snoRNAs suggest that these classes of RNA may have an evolutionary relationship. PMID:19763159

  10. Autoregulation of expression of the yeast Dbp2p 'DEAD-box' protein is mediated by sequences in the conserved DBP2 intron.

    PubMed Central

    Barta, I; Iggo, R

    1995-01-01

    The human p68, Saccharomyces cerevisiae DBP2 and Schizosaccharomyces pombe dbp2 genes are closely related members of the 'DEAD-box' RNA helicase superfamily. All three genes contain an intron at a conserved site in RNA helicase motif V. The S.cerevisiae intron is unusual both for its position near the 3'-end of the open reading frame and for its size, 1001 nucleotides. We show here that precise deletion of the intron has no effect on cell viability but leads to an increase in Dbp2p protein expression. Inefficient splicing due to the size of the intron can not account for this difference because the intron is efficiently spliced in Dbp2p-deficient cells. Instead, there is a reciprocal relationship between the amount of Dbp2p in the cell and the efficiency with which DBP2 intron-containing genes are expressed. Inactive Dbp2p mutants are efficiently expressed from DBP2 intron-containing plasmids, and fragments of the DBP2 intron confer Dbp2p-responsiveness on heterologous reporter introns. This suggest that there is an intron-mediated negative feedback loop regulating DBP2 expression, and provides a possible explanation for the retention of such an unusual intron in S.cerevisiae. Images PMID:7641698

  11. C/D box sRNA, CRISPR RNA and tRNA processing in an archaeon with a minimal fragmented genome.

    PubMed

    Richter, Hagen; Mohr, Sabine; Randau, Lennart

    2013-02-01

    The analysis of deep sequencing data allows for a genome-wide overview of all the small RNA molecules (the 'sRNome') that are present in a single organism. In the present paper, we review the processing of CRISPR (clustered regularly interspaced short palindromic repeats) RNA, C/D box sRNA (small non-coding RNA) and tRNA in Nanoarchaeum equitans. The minimal and fragmented genome of this tiny archaeon permits a sequencing depth that enables the identification of processing intermediates in the study of RNA processing pathways. These intermediates include circular C/D box sRNA molecules and tRNA half precursors.

  12. The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

    PubMed

    Dubey, P; Hendrickson, R C; Meredith, S C; Siegel, C T; Shabanowitz, J; Skipper, J C; Engelhard, V H; Hunt, D F; Schreiber, H

    1997-02-17

    The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

  13. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

    PubMed Central

    Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G.; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I.; Hentschke, Moritz; Aepfelbacher, Martin

    2016-01-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  14. The Immunodominant Antigen of an Ultraviolet-induced Regressor Tumor Is Generated by a Somatic Point Mutation in the DEAD Box Helicase p68

    PubMed Central

    Dubey, Purnima; Hendrickson, Ronald C.; Meredith, Stephen C.; Siegel, Christopher T.; Shabanowitz, Jeffrey; Skipper, Jonathan C.A.; Engelhard, Victor H.; Hunt, Donald F.; Schreiber, Hans

    1997-01-01

    The genetic origins of CD8+ T cell–recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A− 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor–specific mutation in the coding region of a nucleic acid helicase. PMID:9034148

  15. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells.

    PubMed

    Berneking, Laura; Schnapp, Marie; Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I; Hentschke, Moritz; Aepfelbacher, Martin

    2016-06-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  16. Identification of the human DEAD-box protein p68 as a substrate of Tlk1

    SciTech Connect

    Kodym, Reinhard . E-mail: reinhard.kodym@meduniwien.ac.at; Henoeckl, Christian; Fuerweger, Christoph

    2005-07-29

    The activity of the human protein kinase Tlk1 is down-regulated within minutes after exposure of cells to ionizing radiation. In order to identify signaling pathways which might be relevant in the radiation response of mammalian cells we screened nuclear proteins for substrates of Tlk1. Among several proteins one could be identified as p68 RNA helicase. Furthermore, it could be shown that Tlk1 phosphorylates immunoprecipitated p68. The phosphorylation of the C-terminal fragment of p68 by rTlk1 reduced its affinity to single stranded RNA in a gel shift assay. In addition, it could be demonstrated that increasing the Tlk1 activity in HT1080 cells by forced Tlk1 overexpression leads to an increased phosphorylation of endogenous p68, arguing that p68 might be a physiological substrate of Tlk1. These findings open the possibility that Tlk1 might participate in diverse biologic functions like cell growth and differentiation, pre-mRNA splicing, and transcriptional coactivation.

  17. MicroRNA regulation of F-box proteins and its role in cancer.

    PubMed

    Wu, Zhao-Hui; Pfeffer, Lawrence M

    2016-02-01

    MicroRNAs (miRNAs) are small endogenous non-coding RNAs, which play critical roles in cancer development by suppressing gene expression at the post-transcriptional level. In general, oncogenic miRNAs are upregulated in cancer, while miRNAs that act as tumor suppressors are downregulated, leading to decreased expression of tumor suppressors and upregulated oncogene expression, respectively. F-box proteins function as the substrate-recognition components of the SKP1-CUL1-F-box (SCF)-ubiquitin ligase complex for the degradation of their protein targets by the ubiquitin-proteasome system. Therefore F-box proteins and miRNAs both negatively regulate target gene expression post-transcriptionally. Since each miRNA is capable of fine-tuning the expression of multiple target genes, multiple F-box proteins may be suppressed by the same miRNA. Meanwhile, one F-box proteins could be regulated by several miRNAs in different cancer types. In this review, we will focus on miRNA-mediated downregulation of various F-box proteins, the resulting stabilization of F-box protein substrates and the impact of these processes on human malignancies. We provide insight into how the miRNA: F-box protein axis may regulate cancer progression and metastasis. We also consider the broader role of F-box proteins in the regulation of pathways that are independent of the ubiquitin ligase complex and how that impacts on oncogenesis. The area of miRNAs and the F-box proteins that they regulate in cancer is an emerging field and will inform new strategies in cancer treatment.

  18. The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

    PubMed Central

    Granneman, Sander; Zhu, Jieyi; Gill, Michael; Papoulas, Ophelia; Marcotte, Edward M.; Tollervey, David; Correll, Carl C.; Johnson, Arlen W.

    2015-01-01

    In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins. PMID:25710520

  19. The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.

    PubMed

    Sardana, Richa; Liu, Xin; Granneman, Sander; Zhu, Jieyi; Gill, Michael; Papoulas, Ophelia; Marcotte, Edward M; Tollervey, David; Correll, Carl C; Johnson, Arlen W

    2015-02-01

    In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.

  20. Single-molecule studies reveal that DEAD box protein DDX1 promotes oligomerization of HIV-1 Rev on the Rev response element.

    PubMed

    Robertson-Anderson, Rae M; Wang, Jun; Edgcomb, Stephen P; Carmel, Andrew B; Williamson, James R; Millar, David P

    2011-07-29

    Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev-RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev-RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to nonproductive nucleation events, resulting in increased occurrence of higher-order Rev-RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (adenylyl-imidophosphate), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and adenylyl-imidophosphate, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE. PMID:21763499

  1. Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells

    PubMed Central

    Stepanov, Grigoriy A.; Semenov, Dmitry V.; Savelyeva, Anna V.; Kuligina, Elena V.; Koval, Olga A.; Rabinov, Igor V.; Richter, Vladimir A.

    2013-01-01

    Box C/D small nucleolar RNAs (snoRNAs) are known to guide the 2′-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct 2′-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes. PMID:23607094

  2. Factors that influence T box riboswitch efficacy and tRNA affinity.

    PubMed

    Zeng, C; Zhou, S; Bergmeier, S C; Hines, J V

    2015-09-01

    The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors. PMID:26220520

  3. Importance and determinants of induction of cold-induced DEAD RNA helicase in the hyperthermophilic archaeon Thermococcus kodakarensis.

    PubMed

    Nagaoka, Eriko; Hidese, Ryota; Imanaka, Tadayuki; Fujiwara, Shinsuke

    2013-08-01

    Thermococcus kodakarensis, which grows optimally at 85°C, expresses cold stress-inducible DEAD box RNA helicase (Tk-deaD) when shifted to 60°C. A DDA1 deletion (ΔTk-deaD) mutant exhibited decreased cell growth, and cells underwent lysis at 60°C in nutrient broth in the absence of elemental sulfur. In contrast, cells in medium containing elemental sulfur at 60°C did not undergo lysis, suggesting that Tk-deaD is necessary for cell growth in sulfur-free medium. To identify the element responsible for the cold response, a pTKR expression probe plasmid was constructed using thermostable catalase from Pyrobaculum calidifontis as a reporter. The plasmid pTKRD, which contained the transcription factor B recognition element, TATA region, and Shine-Dalgarno (SD) region, including the initiation codon of the Tk-deaD gene, exhibited cold inducibility. We also constructed a series of deletion and chimeric constructs with the glutamate dehydrogenase (gdh) promoter, whose expression is constitutive independent of culture temperatures and catalase expression. Reporter assay experiments indicated that the regulatory element is located in the region between the SD region and the initiation codon (ATG). Nucleotide sequences in the upstream regions of Tk-deaD and gdh were compared and revealed a five-adenosine (AAAAA) sequence between SD and ATG of Tk-deaD that was not present in gdh. Replacement of the repeated adenosine sequence with other sequences revealed that the AAAAA sequence is important for cold induction. This sequence-specific mechanism is unique and is one that has not been identified in other known cold-inducible genes. PMID:23729644

  4. Importance and Determinants of Induction of Cold-Induced DEAD RNA Helicase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

    PubMed Central

    Nagaoka, Eriko; Hidese, Ryota; Imanaka, Tadayuki

    2013-01-01

    Thermococcus kodakarensis, which grows optimally at 85°C, expresses cold stress-inducible DEAD box RNA helicase (Tk-deaD) when shifted to 60°C. A DDA1 deletion (ΔTk-deaD) mutant exhibited decreased cell growth, and cells underwent lysis at 60°C in nutrient broth in the absence of elemental sulfur. In contrast, cells in medium containing elemental sulfur at 60°C did not undergo lysis, suggesting that Tk-deaD is necessary for cell growth in sulfur-free medium. To identify the element responsible for the cold response, a pTKR expression probe plasmid was constructed using thermostable catalase from Pyrobaculum calidifontis as a reporter. The plasmid pTKRD, which contained the transcription factor B recognition element, TATA region, and Shine-Dalgarno (SD) region, including the initiation codon of the Tk-deaD gene, exhibited cold inducibility. We also constructed a series of deletion and chimeric constructs with the glutamate dehydrogenase (gdh) promoter, whose expression is constitutive independent of culture temperatures and catalase expression. Reporter assay experiments indicated that the regulatory element is located in the region between the SD region and the initiation codon (ATG). Nucleotide sequences in the upstream regions of Tk-deaD and gdh were compared and revealed a five-adenosine (AAAAA) sequence between SD and ATG of Tk-deaD that was not present in gdh. Replacement of the repeated adenosine sequence with other sequences revealed that the AAAAA sequence is important for cold induction. This sequence-specific mechanism is unique and is one that has not been identified in other known cold-inducible genes. PMID:23729644

  5. Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

    PubMed

    Filippova, J A; Stepanov, G A; Semenov, D V; Koval, O A; Kuligina, E V; Rabinov, I V; Richter, V A

    2015-01-01

    Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans. PMID:26085946

  6. Binding of Y-box proteins to RNA: involvement of different protein domains.

    PubMed Central

    Ladomery, M; Sommerville, J

    1994-01-01

    Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA. Images PMID:7530842

  7. The T box mechanism: tRNA as a regulatory molecule

    PubMed Central

    Green, Nicholas J.; Grundy, Frank J.; Henkin, Tina M.

    2009-01-01

    The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. PMID:19932103

  8. Box C/D sRNA stem ends act as stabilizing anchors for box C/D di-sRNPs

    PubMed Central

    Yip, W. S. Vincent; Shigematsu, Hideki; Taylor, David W.; Baserga, Susan J.

    2016-01-01

    Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′-O-methylation, one rRNA modification type in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs have demonstrated the dimeric sRNP (di-sRNP) architecture, which has been corroborated by nuclear magnetic resonance (NMR) studies. Due to limitations of the structural techniques, the orientation of the box C/D sRNAs has remained unclear. Here, we have used cryo-EM to elucidate the sRNA orientation in a M. jannaschii box C/D di-sRNP. The cryo-EM reconstruction suggests a parallel orientation of the two sRNAs. Biochemical and structural analyses of sRNPs assembled with mutant sRNAs indicate a potential interaction between the sRNA stem ends. Our results suggest that the parallel arrangement of the sRNAs juxtaposes their stem ends into close proximity to allow for a stabilizing interaction that helps maintain the di-sRNP architecture. PMID:27342279

  9. Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box.

    PubMed

    La Teana, A; Brandi, A; O'Connor, M; Freddi, S; Pon, C L

    2000-10-01

    The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.

  10. Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis.

    PubMed

    Germain, Devon R; Li, Lei; Hildebrandt, Matthew R; Simmonds, Andrew J; Hughes, Sarah C; Godbout, Roseline

    2015-11-15

    Mammalian DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. Here, we report phenotypes associated with a null Ddx1 (Ddx1(AX)) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. We demonstrate that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. Our data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing.

  11. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers

    PubMed Central

    Zhang, Jinwei; Ferré-DAmaré, Adrian R.

    2016-01-01

    Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA) and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers. PMID:27043647

  12. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers.

    PubMed

    Zhang, Jinwei; Ferré-DAmaré, Adrian R

    2016-01-01

    Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA) and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers. PMID:27043647

  13. Divergent roles of the DEAD-box protein BS-PL10, the urochordate homologue of human DDX3 and DDX3Y proteins, in colony astogeny and ontogeny.

    PubMed

    Rosner, Amalia; Paz, Guy; Rinkevich, Baruch

    2006-06-01

    Proteins of the highly conserved PL-10 (Ded1P) subfamily of DEAD-box family, participate in a wide variety of biological functions. However, the entire spectrum of their functions in both vertebrates and invertebrates is still unknown. Here, we isolated the Botryllus schlosseri (Urochordata) homologue, BS-PL10, revealing its distributions and functions in ontogeny and colony astogeny. In botryllid ascidians, the colony grows by increasing the number of modular units (each called a zooid) through a whole colony synchronized and weekly cyclical astogenic budding process (blastogenesis). At the level of the colony, both BS-PL10 mRNA and its protein (78 kDa) fluctuate in a weekly pattern that corresponds with the animal's blastogenic cycle, increasing from blastogenic stage A to blastogenic stage D. At the organ/module level, a sharp decline is revealed. Primary and secondary developing buds express high levels of BS-PL10 mRNA and protein at all blastogeneic stages. These levels are reduced four to nine times in the new set of functional zooids. This portrait of colony astogeny differed from its ontogeny. Oocytes and sperm cells express high levels of BS-PL10 protein only at early stages of development. Young embryos reveal background levels with increased expressions in some organs at more developed stages. Results reveal that higher levels of BS-PL10 mRNA and protein are characteristic to multipotent soma and germ cells, but patterns deviate between two populations of differentiating stem cells, the stem cells involved in weekly blastogenesis and stem cells involved in embryogenesis. Two types of experimental manipulations, zooidectomy and siRNA assays, have confirmed the importance of BS-PL10 for cell differentiation and organogenesis. BS-PL10 (phylogenetically matching the animal's position in the evolutionary tree), is the only member of this subfamily in B. schlosseri, featuring a wide range of biological activities, some of which represent pivotal roles. The

  14. Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

    PubMed

    Xiao, Xingqing; Hung, Michelle E; Leonard, Joshua N; Hall, Carol K

    2016-10-15

    Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

  15. Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase.

    PubMed

    Li, Shuang; Duan, Jingqi; Li, Dandan; Yang, Bing; Dong, Mengqiu; Ye, Keqiong

    2011-11-15

    Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.

  16. Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing.

    PubMed

    Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta; Hidmi, Sana; Agranat-Tamir, Lily; Korotkov, Konstantin; Nevo, Yuval; Eyras, Eduardo; Sperling, Ruth; Stamm, Stefan

    2016-03-22

    C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes. PMID:26957605

  17. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    PubMed

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product. PMID:26925607

  18. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    PubMed

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.

  19. Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

    PubMed Central

    Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta; Hidmi, Sana; Agranat-Tamir, Lily; Korotkov, Konstantin; Nevo, Yuval; Sperling, Ruth; Stamm, Stefan

    2016-01-01

    C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA–RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously “silent” exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes. PMID:26957605

  20. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

    PubMed Central

    Kiledjian, M; Dreyfuss, G

    1992-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity. Images PMID:1628625

  1. T box transcription antitermination riboswitch: Influence of nucleotide sequence and orientation on tRNA binding by the antiterminator element

    PubMed Central

    Fauzi, Hamid; Agyeman, Akwasi; Hines, Jennifer V.

    2008-01-01

    Many bacteria utilize riboswitch transcription regulation to monitor and appropriately respond to cellular levels of important metabolites or effector molecules. The T box transcription antitermination riboswitch responds to cognate uncharged tRNA by specifically stabilizing an antiterminator element in the 5′-untranslated mRNA leader region and precluding formation of a thermodynamically more stable terminator element. Stabilization occurs when the tRNA acceptor end base pairs with the first four nucleotides in the seven nucleotide bulge of the highly conserved antiterminator element. The significance of the conservation of the antiterminator bulge nucleotides that do not base pair with the tRNA is unknown, but they are required for optimal function. In vitro selection was used to determine if the isolated antiterminator bulge context alone dictates the mode in which the tRNA acceptor end binds the bulge nucleotides. No sequence conservation beyond complementarity was observed and the location was not constrained to the first four bases of the bulge. The results indicate that formation of a structure that recognizes the tRNA acceptor end in isolation is not the determinant driving force for the high phylogenetic sequence conservation observed within the antiterminator bulge. Additional factors or T box leader features more likely influenced the phylogenetic sequence conservation. PMID:19152843

  2. The BAT1 gene in the MHC encodes an evolutionarily conserved putative nuclear RNA helicase of the DEAD family

    SciTech Connect

    Peelman, L.J.; Van Zeveren, A.; Coppeiters, W.

    1995-03-20

    The BAT1 gene has previously been identified about 30 kb upstream from the tumor necrosis factor (TNF) locus and close to a NF{sub kb}-related gene of the nuclear factor family in the major histocompatibility complex (MHC) of human, mouse, and pig. We now show that the BAT1 translation product is the homolog of the rat p47 nuclear protein, the WM6 Drosophila gene product, and probably also Ce08102 of Caenorhabditis elegans, all members of the DEAD protein family of ATP-dependent RNA helicases. This family has more than 40 members, including the eukaryotic translation initiation factor-4A (eIF-4A), the human nuclear protein p68, and the Drosophila oocyte polar granule component vasa. BAT1 spans about 10 kb, is split into 10 exons of varying length, and encodes a protein of 428 amino acids ({approximately}48 kDa). Human and pig BAT1 cDNAs display 95.6% identity in the coding region and 80% identity in the 5{prime} and 3{prime} noncoding regions. Several repeat sequences of different types were identified in introns of the porcine BAT1 gene. Three different mRNAs, 4.1,1.7, and 0.9 kb, respectively, were detected in all tissues analyzed upon hybridization with porcine BAT1 cDNA. Transfection and expression of human BAT1 cDNA after tagging with a heterologous antibody recognition epitope revealed a nuclear localization of the hybrid protein. An MspI RFLP was detected in an SLA class I typed family, confirming the localization of the BAT1 gene in the porcine MHC. BAT1 thus encodes a putative nuclear ATP-dependent RNA helicase and is likely to have an indispensable function. 35 refs., 6 figs., 1 tab.

  3. DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression

    PubMed Central

    Bäreclev, Caroline; Vaitkevicius, Karolis; Netterling, Sakura; Johansson, Jörgen

    2014-01-01

    RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells. PMID:25590644

  4. Arabidopsis root initiation defective1, a DEAH-box RNA helicase involved in pre-mRNA splicing, is essential for plant development.

    PubMed

    Ohtani, Misato; Demura, Taku; Sugiyama, Munetaka

    2013-06-01

    Pre-mRNA splicing is a critical process in gene expression in eukaryotic cells. A multitude of proteins are known to be involved in pre-mRNA splicing in plants; however, the physiological roles of only some of these have been examined. Here, we investigated the developmental roles of a pre-mRNA splicing factor by analyzing root initiation defective1-1 (rid1-1), an Arabidopsis thaliana mutant previously shown to have severe defects in hypocotyl dedifferentiation and de novo meristem formation in tissue culture under high-temperature conditions. Phenotypic analysis in planta indicated that RID1 is differentially required during development and has roles in processes such as meristem maintenance, leaf morphogenesis, and root morphogenesis. RID1 was identified as encoding a DEAH-box RNA helicase implicated in pre-mRNA splicing. Transient expression analysis using intron-containing reporter genes showed that pre-mRNA splicing efficiency was affected by the rid1 mutation, which supported the presumed function of RID1 in pre-mRNA splicing. Our results collectively suggest that robust levels of pre-mRNA splicing are critical for several specific aspects of plant development.

  5. Assembly into snoRNP controls 5'-end maturation of a box C/D snoRNA in Saccharomyces cerevisiae

    SciTech Connect

    Preti, Milena; Guffanti, Elisa; Valitutto, Eleonora; Dieci, Giorgio . E-mail: giorgio.dieci@unipr.it

    2006-12-15

    The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5'-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5'-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3'-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Our data suggest a functional coupling between snoRNP assembly and 5'-end maturation of independently transcribed box C/D snoRNAs.

  6. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  7. Optimizations of siRNA design for the activation of gene transcription by targeting the TATA-box motif.

    PubMed

    Fan, Miaomiao; Zhang, Yijun; Huang, Zhuoqiong; Liu, Jun; Guo, Xuemin; Zhang, Hui; Luo, Haihua

    2014-01-01

    Small interfering RNAs (siRNAs) are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs) could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a) complementary to the TATA-box-centered region; (b) UA usage at the first two bases of the antisense strand; (c) twenty-three nucleotides (nts) in length; (d) 2'-O-Methyl (2'-OMe) modification at the 3' terminus of the antisense strand; (e) avoiding mismatches at the 3' end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2) gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.

  8. Differential roles of archaeal box H/ACA proteins in guide RNA-dependent and independent pseudouridine formation.

    PubMed

    Gurha, Priyatansh; Joardar, Archi; Chaurasia, Priyasri; Gupta, Ramesh

    2007-10-01

    RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55.

  9. Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation.

    PubMed

    Yang, Xinxing; Duan, Jingqi; Li, Shuang; Wang, Peng; Ma, Shoucai; Ye, Keqiong; Zhao, Xin Sheng

    2012-11-01

    The box H/ACA RNA-guided pseudouridine synthase is a complicated ribonucleoprotein enzyme that recruits substrate via both the guide RNA and the catalytic subunit Cbf5. Structural studies have revealed multiple conformations of the enzyme, but a quantitative description of the reaction pathway is still lacking. Using fluorescence correlation spectroscopy, we here measured the equilibrium dissociation constants and kinetic association and dissociation rates of substrate and product complexes mimicking various reaction intermediate states. These data support a sequential model for substrate loading and product release regulated by the thumb loop of Cbf5. The uridine substrate is first bound primarily through interaction with the guide RNA and then loaded into the active site while progressively interacted with the thumb. After modification, the subtle chemical structure change from uridine to pseudouridine at the target site triggers the release of the thumb, resulting in an intermediate complex with the product bound mainly by the guide RNA. By dissecting the role of Gar1 in individual steps of substrate turnover, we show that Gar1 plays a major role in catalysis and also accelerates product release about 2-fold. Our biophysical results integrate with previous structural knowledge into a coherent reaction pathway of H/ACA RNA-guided pseudouridylation.

  10. Emerging from the clouds: Vasa helicase sheds light on piRNA amplification.

    PubMed

    Claycomb, Julie M

    2014-06-23

    Reporting in a recent study in Cell, Xiol and colleagues (2014) identify the conserved DEAD box helicase Vasa as a platform for secondary Piwi-interacting RNA (piRNA) biogenesis in the insect nuage. This study represents a substantial breakthrough in understanding the mechanism and location of piRNA amplification by the "ping-pong" cycle.

  11. The Conserved FRNK Box in HC-Pro, a Plant Viral Suppressor of Gene Silencing, Is Required for Small RNA Binding and Mediates Symptom Development▿ †

    PubMed Central

    Shiboleth, Yoel Moshe; Haronsky, Elina; Leibman, Diana; Arazi, Tzahi; Wassenegger, Michael; Whitham, Steven A.; Gaba, Victor; Gal-On, Amit

    2007-01-01

    The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMVFRNK caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA “passenger” strands (miRNA*s), in systemically infected leaves than the attenuated ZYMVFINK did. HC-ProFRNK specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-ProFINK. Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HCFINK mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development. PMID:17898058

  12. Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis

    PubMed Central

    Enwerem, Isioma I.; Velma, Venkatramreddy; Broome, Hanna J.; Kuna, Marija; Begum, Rowshan A.; Hebert, Michael D.

    2014-01-01

    ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase. PMID:24659245

  13. Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

    PubMed

    Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed; Santiago, Ace George; Chen, Tai-Yen; Jung, Won; Helmann, John D; Chen, Peng

    2015-11-01

    Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

  14. Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

    PubMed

    Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed; Santiago, Ace George; Chen, Tai-Yen; Jung, Won; Helmann, John D; Chen, Peng

    2015-11-01

    Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription. PMID:26483469

  15. The asparagine residue in the FRNK box of potyviral helper-component protease is critical for its small RNA binding and subcellular localization.

    PubMed

    Sahana, Nandita; Kaur, Harpreet; Jain, R K; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

    2014-05-01

    The multifunctional potyviral helper-component protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at the FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variants for the N(182) residue are known to occur. Here, a mutant at HcPro(N182L) was developed to investigate its role in natural populations. Using in vitro studies, we found an increase in the small RNA (sRNA) binding potential of HcPro(N182L) without affecting its protein-protein interaction properties, suggesting that the presence of N(182) is critical to maintain threshold levels of sRNAs, but does not interfere in the self-interaction of HcPro. Furthermore, we found that expression of HcPro(N182L) in Nicotiana benthamiana affected plant growth. Transient expression of HcPro(N182L) induced reporter gene expression in 16c GFP transgenic plants more than HcPro did, suggesting that replacement of asparagine in the FRNK box favours RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcPro(N182L) was observed only in cytoplasmic inclusion bodies in N. benthamiana leaves, when fused to a GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerization of HcPro. These findings suggest that amino acid N(182) of the conserved FRNK box may regulate RNA silencing mechanisms, and is required for maintenance of the subcellular localization of the protein for its multi-functionality. Hence, the N(182) residue of the FRNK box seems to be indispensable for potyvirus infection during evolution. PMID:24526574

  16. microRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development.

    PubMed

    Ferreira e Silva, Geraldo Felipe; Silva, Eder Marques; Azevedo, Mariana da Silva; Guivin, Mike Anderson Corazon; Ramiro, Daniel Alves; Figueiredo, Cassia Regina; Carrer, Helaine; Peres, Lázaro Eustáquio Pereira; Nogueira, Fabio Tebaldi Silveira

    2014-05-01

    Fruit ripening in tomato (Solanum lycopersicum L.) is well understood at the molecular level. However, information regarding genetic pathways associated with tomato ovary and early fruit development is still lacking. Here, we investigate the possible role(s) of the microRNA156/SQUAMOSA promoter-binding protein-like (SPL or SBP box) module (miR156 node) in tomato ovary development. miR156-targeted S. lycopersicum SBP genes were dynamically expressed in developing flowers and ovaries, and miR156 was mainly expressed in meristematic tissues of the ovary, including placenta and ovules. Transgenic tomato cv. Micro-Tom plants over-expressing the AtMIR156b precursor exhibited abnormal flower and fruit morphology, with fruits characterized by growth of extra carpels and ectopic structures. Scanning electron microscopy and histological analyses showed the presence of meristem-like structures inside the ovaries, which are probably responsible for the ectopic organs. Interestingly, expression of genes associated with meristem maintenance and formation of new organs, such as LeT6/TKN2 (a KNOX-like class I gene) and GOBLET (a NAM/CUC-like gene), was induced in developing ovaries of transgenic plants as well as in the ovaries of the natural mutant Mouse ear (Me), which also displays fruits with extra carpels. Conversely, expression of the MADS box genes MACROCALYX (MC) and FUL1/TDR4, and the LEAFY ortholog FALSIFLORA, was repressed in the developing ovaries of miR156 over-expressors, suggesting similarities with Arabidopsis at this point of the miR156/SPL pathway but with distinct functional consequences in reproductive development. Altogether, these observations suggest that the miR156 node is involved in maintenance of the meristematic state of ovary tissues, thereby controlling initial steps of fleshy fruit development and determinacy.

  17. Upregulation of microRNA-370 facilitates the repair of amputated fingers through targeting forkhead box protein O1

    PubMed Central

    Zhang, Hongxing; Sun, Xiaojuan

    2015-01-01

    Angiogenesis is critical to the success of digital replantation. Recent study suggests an important regulatory role of microRNA-370 (miR-370) in ischemia–reperfusion injury. However, its function in digital replantation is poorly understood. In this study, we reported that the expression of miR-370 was upregulated in replantation tissues. miR-370 mimic transfection promoted human umbilical vein endothelial cells (HUVECs) proliferation by regulating the cell cycle and inhibited apoptosis. miR-370 mimic transfection also significantly increased HUVECs migration and induced the formation of capillary-like structures in HUVECs, indicated that miR-370 promoted capillary tube formation in vitro. Furthermore, forkhead box protein O1 (FOXO1) was identified as the functional target of miR-370 by dual-luciferase reporter assay. FOXO1 overexpression vector lacked 3′-UTR together with miR-370 mimic transfection strongly abrogated miR-370-induced cell proliferation and the formation of capillary-like structures in HUVECs. Taken together, our results revealed that the upregulation of miR-370 might facilitate the repair of amputated fingers by regulating angiogenesis through targeting FOXO1. This study provided a potential therapeutic target for the restoration of finger function after replantation. PMID:26316586

  18. Non-Conserved Residues in Clostridium acetobutylicum tRNAAla Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch

    PubMed Central

    Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. PMID:26426057

  19. Association of genetic polymorphism of pre-microRNA-146a rs2910164 and serum high-mobility group box 1 with febrile seizures in Egyptian children.

    PubMed

    Issac, Marianne Samir Makboul; Girgis, Marian; Haroun, Mervat; Shalaby, Amal

    2015-03-01

    Interaction between immune-inflammatory process and genetic factors might be implicated in the pathogenesis of febrile seizures. Pre-microRNA (miR)-146a rs2910164 polymorphism is postulated to modulate expression of miR-146a whose anti-inflammatory role involves regulation of high-mobility group box 1. Our aim is to examine whether rs2910164 polymorphism influences serum high-mobility group box 1 levels and whether an association exists between both and febrile seizures. The study included 136 children, divided into 4 groups. Real-time polymerase chain reaction was used for detection of rs2910164 polymorphism and high-mobility group box 1 was measured using enzyme-linked immunosorbent assay. High-mobility group box 1 levels were higher in febrile seizure patients compared to the other groups. Rs2910164 polymorphism was not associated with increased risk of febrile seizures. Rs2910164 polymorphism might be accompanied by an upregulation of the proinflammatory process as it might be associated with an increase in high-mobility group box 1 and leukocytic count.

  20. Jeweled Boxes

    ERIC Educational Resources Information Center

    Coy, Mary

    2009-01-01

    While an empty cardboard box from a ream of copy paper may be the most coveted box among teachers in the author's school, for other people, brass boxes from India, Khokhlova lacquer boxes from Russia, and puzzle boxes from Japan are more the type that are collected and admired. Whether it is used for storage or decoration, a box can evoke a sense…

  1. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine

    PubMed Central

    Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Chai, Yu-Shuang; Wang, Yu-Gang; Jiang, Jing-Fei; Feng, Tian-Shi; Wang, Xin-Pei; Yu, Xuan; Yan, Xiao-Jin; Xing, Dong-Ming; Du, Li-Jun

    2015-01-01

    Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine’s pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies. PMID:26671652

  2. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry.

    PubMed

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C; Liu, Zhongchi

    2015-09-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants. PMID:26143249

  3. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry.

    PubMed

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C; Liu, Zhongchi

    2015-09-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants.

  4. A truncated hnRNP A1 isoform, lacking the RGG-box RNA binding domain, can efficiently regulate HIV-1 splicing and replication.

    PubMed

    Jean-Philippe, Jacques; Paz, Sean; Lu, Michael L; Caputi, Massimo

    2014-01-01

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is one of the most abundant RNA binding proteins. hnRNP A1 is localized prevalently in the nucleus but it can relocate to the cytoplasm in response to specific stimuli shuttling between nuclear and cytoplasmic compartments. The cellular localization of this protein is regulated by a short C-terminus motif (M9) and other less defined sequences. The RNA binding specificity of this protein is dependent on multiple RNA binding domains (RBDs), which regulate its role in RNA processing and expression. hnRNP A1 plays multiple roles in gene expression by regulating the biogenesis and translation of messengers RNAs, the processing of miRNAs, affecting transcription and controlling telomere maintenance. The multiple functions of this protein correlate with diverse roles in genetic disease, cancer and the replication of viral pathogens. Utilizing a tagged hnRNP A1 deletion library we have shown that the three hnRNP A1 RBDs contribute to the prevalent nuclear distribution of the protein. Our data also indicate that a truncated form of the protein, lacking one of the RBDs, the RGG-box, can regulate splicing of a splicing reporter minigene and down-regulate replication of the HIV-1 virus with efficiency comparable to the wild-type protein. This functional hnRNP A1 deletion mutant is similar to a predicted hnRNP A1 isoform, which had not been previously experimentally characterized. PMID:24530421

  5. Chicken Y-box proteins chk-YB-1b and chk-YB-2 repress translation by sequence-specific interaction with single-stranded RNA.

    PubMed Central

    Swamynathan, S K; Nambiar, A; Guntaka, R V

    2000-01-01

    Y-Box proteins comprise a large family of multifunctional proteins with a wide spectrum of activities in both transcription and translational regulation of gene expression. Earlier, we have reported on the involvement of chk-YB-2 in transcriptional regulation of Rous sarcoma virus long terminal repeats and the involvement of chk-YB-1b in transcriptional regulation of alpha1(I) collagen genes. Here, we have investigated the potential role of chk-YB-2 and chk-YB-1b in RNA metabolism. We report that chk-YB-2 and chk-YB-1b are localized predominantly in the cytoplasm and that they both can bind single-stranded RNA in a sequence-specific and reversible manner. Well-conserved cold-shock domain, N-terminal proline-rich domain and the alternating clusters of acidic and basic amino acids located in the C-terminal ends of these two proteins were all found to be necessary for their RNA-binding ability. Further, we demonstrate that these two proteins inhibit translation in vitro and that binding to RNA is required for this inhibition. The significance of these results is discussed. PMID:10816422

  6. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments.

  7. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  8. RNA size is a critical factor for U-containing substrate selectivity and permanent pseudouridylated product release during the RNA:Ψ-synthase reaction catalyzed by box H/ACA sRNP enzyme at high temperature.

    PubMed

    Tillault, Anne-Sophie; Fourmann, Jean-Baptiste; Loegler, Christine; Blaud, Magali; Branlant, Christiane; Charpentier, Bruno

    2015-06-01

    The box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Ψ) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyperthermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to Ψ conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity.

  9. Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

    PubMed Central

    Kumar P, Pavan; Emechebe, Uchenna; Smith, Richard; Franklin, Sarah; Moore, Barry; Yandell, Mark; Lessnick, Stephen L; Moon, Anne M

    2014-01-01

    Cellular senescence is a crucial tumor suppressor mechanism. We discovered a CAPERα/TBX3 repressor complex required to prevent senescence in primary cells and mouse embryos. Critical, previously unknown roles for CAPERα in controlling cell proliferation are manifest in an obligatory interaction with TBX3 to regulate chromatin structure and repress transcription of CDKN2A-p16INK and the RB pathway. The IncRNA UCA1 is a direct target of CAPERα/TBX3 repression whose overexpression is sufficient to induce senescence. In proliferating cells, we found that hnRNPA1 binds and destabilizes CDKN2A-p16INK mRNA whereas during senescence, UCA1 sequesters hnRNPA1 and thus stabilizes CDKN2A-p16INK. Thus CAPERα/TBX3 and UCA1 constitute a coordinated, reinforcing mechanism to regulate both CDKN2A-p16INK transcription and mRNA stability. Dissociation of the CAPERα/TBX3 co-repressor during oncogenic stress activates UCA1, revealing a novel mechanism for oncogene-induced senescence. Our elucidation of CAPERα and UCA1 functions in vivo provides new insights into senescence induction, and the oncogenic and developmental properties of TBX3. DOI: http://dx.doi.org/10.7554/eLife.02805.001 PMID:24876127

  10. Formation of C-terminally truncated version of the Taz1 protein employs cleavage-box structure in mRNA

    SciTech Connect

    Gunisova, Stanislava; Bartosova, Zdenka; Kramara, Juraj; Nosek, Jozef; Tomaska, Lubomir

    2010-02-12

    When expressed in various hosts the taz1{sup +} gene encoding the fission yeast telomere-binding protein produces two forms of polypeptides: full-length (Taz1p) and truncated (Taz1p{Delta}C) version lacking almost entire Myb-domain. Whereas Taz1p binds telomeric DNA in vitro, Taz1p{Delta}C forms long filaments unable of DNA binding. The formation of Taz1p{Delta}C is a result of neither site-specific proteolysis, nor premature termination of transcription. In silico analysis of the taz1{sup +} RNA transcript revealed a stem-loop structure at the site of cleavage (cleavage box; CB). In order to explore whether it possesses inherent destabilizing effects, we cloned CB sequence into the open reading frame (ORF) of glutathione-S-transferase (GST) and observed that when expressed in Escherichia coli the engineered gene produced two forms of the reporter protein. The formation of the truncated version of GST was abolished, when CB was replaced with recoded sequence containing synonymous codons thus indicating that the truncation is based on structural properties of taz1{sup +} mRNA.

  11. Trilayer micelles for combination delivery of rapamycin and siRNA targeting Y-box binding protein-1 (siYB-1)

    PubMed Central

    Zeng, San; Xiong, May P.

    2013-01-01

    A three layer (trilayer) polymeric micelle system based on the self-association of the triblock polymer poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide}-b-poly(ε-caprolactone) (PEG-b-PAsp(DET)-b-PCL) has been synthesized and investigated for combination delivery of rapamycin (RAP) and siRNA targeting Y-box binding protein-1 (siYB-1). The trilayer micelle is composed of (a) a hydrophilic poly(ethylene glycol) (PEG) block constituting the outer layer to improve pharmacokinetics, (b) an intermediate compartment composed of the cationic poly{2-[(2-aminoethyl)amino] ethyl aspartamide} (PAsp(DET)) segment for interacting with siYB-1, and (c) an inner hydrophobic poly(ε-caprolactone) (PCL) compartment for encapsulation of RAP. A major advantage of this system is biocompatibility since PEG and PCL are both approved by the FDA, and PAsp(DET) is a non-toxic pH responsive cationic poly(amino acid)-based polymer. In this study, it has been shown that PCL can encapsulate RAP with high loading efficiencies, and PAsp(DET) can successfully interact with siRNA for efficient transfection/knockdown with negligible cytotoxicity. The enhanced therapeutic efficacy of RAP/ siYB-1 micelles was demonstrated in cell cultures and in a PC3 xenograft nude mouse model of human prostate cancer. Herein, we demonstrate that trilayer micelles are a promising approach to improve the simultaneous delivery of combination siRNA/drug therapies. PMID:23768780

  12. Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa.

    PubMed

    Xu, Ruirui; Zhang, Shizhong; Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  13. Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

    PubMed Central

    d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Gaspin, Christine; Bachellerie, Jean-Pierre

    2001-01-01

    Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2′-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5′ exon but also onto position 39 (universal tRNA numbering) in the 3′ exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon–intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs. PMID:11713301

  14. Bento Boxes

    ERIC Educational Resources Information Center

    Hasio, Cindy

    2010-01-01

    Bento boxes are common objects in Japanese culture, designed to hold enough lunch for one person. They have individual compartments and sometimes multiple tiers for rice, vegetables, and other side dishes. They are made of materials ranging from wood, cloth, aluminum, or plastic. In general, the greater the number of foods, the better the box is…

  15. Boxed In!

    ERIC Educational Resources Information Center

    Stevens, Judith

    2007-01-01

    Early years practitioners know what parents are reminded of every Christmas Day--it does not matter how long families spend carefully selecting the presents, the children are likely to spend longer playing with the empty boxes! Children are fascinated with boxes and practitioners can capitalise on this and support early mathematical development…

  16. [Dreams and the dead].

    PubMed

    Mestre, Claire

    2006-01-01

    In this article, the author examines the issue of dreams and the dead as an essential tool for transcultural psychotherapy and how the dreamlike vision of the dead and its interpretation constitute a turning point in transcultural therapy. Drawing from a clinical example, the author illustrates how the spatial analogy between dreams and the world of the dead has allowed a patient to reconstruct a psychological space severely disturbed by trauma endured.

  17. Film Boxes.

    ERIC Educational Resources Information Center

    Osterer, Irv

    2002-01-01

    Presents an art lesson in which students created three-dimensional designs for 35mm film packages to improve graphic arts learning. Describes how the students examined and created film boxes using QuarkXPress software. (CMK)

  18. Investigating Aquatic Dead Zones

    ERIC Educational Resources Information Center

    Testa, Jeremy; Gurbisz, Cassie; Murray, Laura; Gray, William; Bosch, Jennifer; Burrell, Chris; Kemp, Michael

    2010-01-01

    This article features two engaging high school activities that include current scientific information, data, and authentic case studies. The activities address the physical, biological, and chemical processes that are associated with oxygen-depleted areas, or "dead zones," in aquatic systems. Students can explore these dead zones through both…

  19. Day of the Dead

    ERIC Educational Resources Information Center

    Dann, Tammy; Murphy, Amy

    2012-01-01

    Foreign Language in Elementary School (FLES) teachers in the West Des Moines schools incorporate the Day of the Dead into the fourth grade curriculum each year. The teachers discuss the Day of the Dead celebration at the Art Center, and many ask for volunteers from fourth grade to participate in the event. Student presentations include a wide…

  20. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  1. Cancer-associated mutants of RNA helicase DDX3X are defective in RNA-stimulated ATP hydrolysis

    PubMed Central

    Partridge, Janet F.; Enemark, Eric J.

    2015-01-01

    The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures that suggest the ABL interacts dynamically with ATP and confirm the interaction occurs in solution by NMR chemical shift perturbation (CSP) and isothermal calorimetry (ITC). DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR CSP to show that DDX3X interacts specifically with double-stranded RNA (dsRNA) through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of S. pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma. PMID:25724843

  2. Exploding Boxes

    ERIC Educational Resources Information Center

    Kinney; Jan

    2011-01-01

    How do you teach the "same old, same old" in an interesting and inexpensive way? Art teachers are forever looking for new angles on the good-old elements and principles. And, as budgets tighten, they are trying to be as frugal as possible while still holding their students' attention. Enter exploding boxes! In conceptualizing the three types of…

  3. Dead Sea Scrolls

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A consortium of researchers from Jet Propulsion Laboratory and three other organizations used charged coupled devices (CCDs) and other imaging enhancement technology to decipher previously unreadable portions of the Dead Sea Scrolls. The technique has potentially important implications for archeology.

  4. ON HYDRODYNAMIC MOTIONS IN DEAD ZONES

    SciTech Connect

    Oishi, Jeffrey S.; Mac Low, Mordecai-Mark E-mail: mordecai@amnh.or

    2009-10-20

    We investigate fluid motions near the midplane of vertically stratified accretion disks with highly resistive midplanes. In such disks, the magnetorotational instability drives turbulence in thin layers surrounding a resistive, stable dead zone. The turbulent layers in turn drive motions in the dead zone. We examine the properties of these motions using three-dimensional, stratified, local, shearing-box, non-ideal, magnetohydrodynamical simulations. Although the turbulence in the active zones provides a source of vorticity to the midplane, no evidence for coherent vortices is found in our simulations. It appears that this is because of strong vertical oscillations in the dead zone. By analyzing time series of azimuthally averaged flow quantities, we identify an axisymmetric wave mode particular to models with dead zones. This mode is reduced in amplitude, but not suppressed entirely, by changing the equation of state from isothermal to ideal. These waves are too low frequency to affect sedimentation of dust to the midplane, but may have significance for the gravitational stability of the resulting midplane dust layers.

  5. "Living versus Dead":

    PubMed Central

    Chakrabarti, Pratik

    2010-01-01

    Summary The Semple antirabies vaccine was developed by David Semple in India in 1911. Semple introduced a peculiarly British approach within the Pasteurian tradition by using carbolized dead virus. This article studies this unique phase of vaccine research between 1910 and 1935 to show that in the debates and laboratory experiments around the potency and safety of vaccines, categories like "living" and "dead" were often used as ideological and moral denominations. These abstract and ideological debates were crucial in defining the final configuration of the Semple vaccine, the most popular antirabies vaccine used globally, and also in shaping international vaccination policies. PMID:21037397

  6. Features of a unique intronless cluster of class I small heat shock protein genes in tandem with box C/D snoRNA genes on chromosome 6 in tomato (Solanum lycopersicum).

    PubMed

    Goyal, Ravinder K; Kumar, Vinod; Shukla, Vijaya; Mattoo, Rohini; Liu, Yongsheng; Chung, Sang Ho; Giovannoni, James J; Mattoo, Autar K

    2012-03-01

    Physical clustering of genes has been shown in plants; however, little is known about gene clusters that have different functions, particularly those expressed in the tomato fruit. A class I 17.6 small heat shock protein (Sl17.6 shsp) gene was cloned and used as a probe to screen a tomato (Solanum lycopersicum) genomic library. An 8.3-kb genomic fragment was isolated and its DNA sequence determined. Analysis of the genomic fragment identified intronless open reading frames of three class I shsp genes (Sl17.6, Sl20.0, and Sl20.1), the Sl17.6 gene flanked by Sl20.1 and Sl20.0, with complete 5' and 3' UTRs. Upstream of the Sl20.0 shsp, and within the shsp gene cluster, resides a box C/D snoRNA cluster made of SlsnoR12.1 and SlU24a. Characteristic C and D, and C' and D', boxes are conserved in SlsnoR12.1 and SlU24a while the upstream flanking region of SlsnoR12.1 carries TATA box 1, homol-E and homol-D box-like cis sequences, TM6 promoter, and an uncharacterized tomato EST. Molecular phylogenetic analysis revealed that this particular arrangement of shsps is conserved in tomato genome but is distinct from other species. The intronless genomic sequence is decorated with cis elements previously shown to be responsive to cues from plant hormones, dehydration, cold, heat, and MYC/MYB and WRKY71 transcription factors. Chromosomal mapping localized the tomato genomic sequence on the short arm of chromosome 6 in the introgression line (IL) 6-3. Quantitative polymerase chain reaction analysis of gene cluster members revealed differential expression during ripening of tomato fruit, and relatively different abundances in other plant parts. PMID:21947620

  7. Dead reckoner navigation project

    NASA Technical Reports Server (NTRS)

    Ellis, R.; Sweet, L.

    1981-01-01

    A previous dead reckoner involved a classical gyrocompass, a Hewlett-Packard minicomputer, and a true airspeed sensor. In an effort to bring the cost of this system more in line with the realities of general aviation, recent work was done on replacing the minicomputer with a microcomputer and implementing a fluidic rate sensor in the compass system in place of the directional gyro.

  8. The Dead Sea

    NASA Technical Reports Server (NTRS)

    2006-01-01

    The Dead Sea is the lowest point on Earth at 418 meters below sea level, and also one of the saltiest bodies of water on Earth with a salinity of about 300 parts-per-thousand (nine times greater than ocean salinity). It is located on the border between Jordan and Israel, and is fed by the Jordan River. The Dead Sea is located in the Dead Sea Rift, formed as a result of the Arabian tectonic plate moving northward away from the African Plate. The mineral content of the Dead Sea is significantly different from that of ocean water, consisting of approximately 53% magnesium chloride, 37% potassium chloride and 8% sodium chloride. In the early part of the 20th century, the Dead Sea began to attract interest from chemists who deduced that the Sea was a natural deposit of potash and bromine. From the Dead Sea brine, Israel and Jordan produce 3.8 million tons potash, 200,000 tons elemental bromine, 45,000 tons caustic soda, 25, 000 tons magnesium metal, and sodium chloride. Both countries use extensive salt evaporation pans that have essentially diked the entire southern end of the Dead Sea.

    With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER images Earth to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18, 1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products.

    The broad spectral coverage and high spectral resolution of ASTER provides scientists in numerous disciplines with critical information for surface mapping, and monitoring of dynamic conditions and temporal change. Example applications are: monitoring glacial advances and retreats; monitoring potentially active volcanoes; identifying crop

  9. The analysis of the inflorescence miRNome of the orchid Orchis italica reveals a DEF-like MADS-box gene as a new miRNA target.

    PubMed

    Aceto, Serena; Sica, Maria; De Paolo, Sofia; D'Argenio, Valeria; Cantiello, Piergiuseppe; Salvatore, Francesco; Gaudio, Luciano

    2014-01-01

    Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the "orchid code" theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs.

  10. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

    PubMed Central

    Jones, R M; Branda, J; Johnston, K A; Polymenis, M; Gadd, M; Rustgi, A; Callanan, L; Schmidt, E V

    1996-01-01

    The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation. PMID:8756633

  11. The RNA Helicase eIF4A Is Required for Sapovirus Translation

    PubMed Central

    Hosmillo, Myra; Sweeney, Trevor R.; Chaudhry, Yasmin; Leen, Eoin; Curry, Stephen

    2016-01-01

    The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation. PMID:26937032

  12. Communicating with the Dead

    NASA Astrophysics Data System (ADS)

    Kurtz, Paul

    2000-03-01

    Communicating with dead persons is popular in the new paranatural (paranormal-spiritual) paradigm that has emerged today. This view violates physicalist principles. Scientists have investigated survival questions for over 150 years. Beginning with the Fox sisters in 1848, they have examined reports of apparitions and ghosts, rappings, table turnings, teleportation, levitation, and other alleged physicalist manifestations in séances. Such reports were discredited by uncovering hoaxes by alleged mediums, e.g. Eusapia Palladino (1910s), Marjorie Crandon (1920s), etc. In recent decades there has been a revival of interest in survival with reports of near-death experiences. Skeptics ask: Are such persons clinically dead? Today, channelers such as James Van Praagh, John Edwards, and Sylvia Browne claim to communicate directly with the dead (the "Sixth Sense"). There is no attempt at physical confirmation or independent eyewitness corroboration so essential for scientific inquiry. Subjective claims are uncritically accepted at their face value, though alternative naturalistic explanations can be given for the alleged phenomena.

  13. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    PubMed

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria. PMID:27412167

  14. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    PubMed

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

  15. UAP56 Couples piRNA Clusters to the Perinuclear Transposon Silencing Machinery

    PubMed Central

    Zhang, Fan; Wang, Jie; Xu, Jia; Zhang, Zhao; Koppetsch, Birgit S.; Schultz, Nadine; Vreven, Thom; Meignin, Carine; Davis, Ilan; Zamore, Phillip D.; Weng, Zhiping; Theurkauf, William E.

    2012-01-01

    Summary piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, while the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 co-localizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope. PMID:23141543

  16. Comparative study of two box H/ACA ribonucleoprotein pseudouridine-synthases: relation between conformational dynamics of the guide RNA, enzyme assembly and activity.

    PubMed

    Fourmann, Jean-Baptiste; Tillault, Anne-Sophie; Blaud, Magali; Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.

  17. Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity

    PubMed Central

    Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21. PMID:23922977

  18. Dead of night.

    PubMed

    Balter, Leon

    2010-07-01

    Dead of Night, the first psychoanalytic horror film, was produced in England in 1945, immediately after the end of World War II--that is, after the English population had suffered systematic Nazi terror from imminent invasion, incessant aerial bombing, and rocket-bombs. This film continued the prewar format of horror films based on themes of the supernatural and the hubris and excesses of science. However, it introduced psychoanalysis as the science in question. The film is structured on two levels: a genteel English country weekend to which witty and urbane guests have been invited; and five horror stories told by the guests. Psychoanalytic insights into this film structure are used here to explain how the film induces horror in the audience.

  19. Dead of night.

    PubMed

    Balter, Leon

    2010-07-01

    Dead of Night, the first psychoanalytic horror film, was produced in England in 1945, immediately after the end of World War II--that is, after the English population had suffered systematic Nazi terror from imminent invasion, incessant aerial bombing, and rocket-bombs. This film continued the prewar format of horror films based on themes of the supernatural and the hubris and excesses of science. However, it introduced psychoanalysis as the science in question. The film is structured on two levels: a genteel English country weekend to which witty and urbane guests have been invited; and five horror stories told by the guests. Psychoanalytic insights into this film structure are used here to explain how the film induces horror in the audience. PMID:20726184

  20. MicroRNA-142-3p inhibits hypoxia/reoxygenation-induced apoptosis and fibrosis of cardiomyocytes by targeting high mobility group box 1

    PubMed Central

    Wang, Yi; Ouyang, Min; Wang, Qiong; Jian, Zaijin

    2016-01-01

    Myocardial ischemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as cardiac fibrosis, which is characterized as the transdifferentiation of fibroblasts to myofibroblasts and collagen deposition. MicroRNAs (miRNAs or miRs) have been demonstrated to be involved in myocardial I/R injury. However, the underlying molecular mechanism remains largely unclear. In the present study, mouse cardiomyocyte M6200 cells were treated with hypoxia/reoxygenation (H/R). Our data indicated that H/R treatment led to cell apoptosis, the increased expression of fibrosis-related proteins, namely collagen I, II, III, and fibronectin, as well as the downregulation of miR-142-3p in M6200 cells. Overexpression of miR-142-3p suppressed the H/R-induced apoptosis and fibrosis of M6200 cells. Bioinformatics analysis and a Dual-Luciferase reporter assay further identified high mobility group box 1 (HMGB1) as a direct target gene of miR-142-3p, and miR-142-3p negatively regulated the protein level of HMGB1 in M6200 cells. Furthermore, knockdown of HMGB1 enhanced cell proliferation whereas it inhibited the apoptosis and fibrosis of M6200 cells. In addition, TGF-β1/Smad3 signaling was suggested to be involved in the miR-142-3p/HMGB1-mediated apoptosis and fibrosis of M6200 cells treated with H/R. Taken together, the findings of the present study demonstrate that miR-142-3p inhibits H/R-induced apoptosis and fibrosis of cardiomyocytes, partly at least, by the direct inhibition of HMGB1 expression. Therefore, these findings have increased our understanding of the pathogenesis of H/R-induced myocardial injury.

  1. MicroRNA-96 promotes the proliferation of colorectal cancer cells and targets tumor protein p53 inducible nuclear protein 1, forkhead box protein O1 (FOXO1) and FOXO3a.

    PubMed

    Gao, Feng; Wang, Wenhui

    2015-02-01

    MicroRNAs (miRNAs) are a conserved class of small, endogenous, non protein-coding RNA molecules that are capable of regulating gene expression at post-transcriptional levels and are involved in diverse cellular processes, including cancer pathogenesis. It has previously been reported that miRNA-96 (miR-96) is overexpressed in human colorectal cancer (CRC). However, the underlying mechanism of miR-96 regulation in CRC remains to be elucidated. In the present study, miR-96 was confirmed to be upregulated in CRC tissues by reverse transcription quantitative polymerase chain reaction. MTT assay, colony formation assay and cell cycle analysis revealed that miR-96 overexpression led to increased tumor cell viability, colony formation ability and cell cycle progression. By contrast, inhibition of miR-96 resulted in the suppression of cell proliferation. It was also demonstrated that miR-96 reduced the messenger RNA and protein expression levels of tumor protein p53 inducible nuclear protein 1 (TP53INP1), forkhead box protein O1 (FOXO1) and FOXO3a, which are closely associated with cell proliferation. A luciferase reporter assay indicated that miR-96 inhibited luciferase intensity controlled by the 3'UTRs of TP53INP1, FOXO1 and FOXO3a. In conclusion, the results of the present study demonstrated that miR-96 contributed to CRC cell growth and that TP53INP1, FOXO1 and FOXO3a were direct targets of miR-96, suggesting that miR-96 may have the potential to be used in the development of miRNA‑based therapies for CRC patients.

  2. New Life From Dead Trees

    ERIC Educational Resources Information Center

    DeGraaf, Richard M.

    1978-01-01

    There are numerous bird species that will nest only in dead or dying trees. Current forestry practices include clearing forests of these snags, or dead trees. This practice is driving many species out of the forests. An illustrated example of bird succession in and on a tree is given. (MA)

  3. Second-site long terminal repeat (LTR) revertants of replication-defective human immunodeficiency virus: effects of revertant TATA box motifs on virus infectivity, LTR-directed expression, in vitro RNA synthesis, and binding of basal transcription factors TFIID and TFIIA.

    PubMed Central

    Kashanchi, F; Shibata, R; Ross, E K; Brady, J N; Martin, M A

    1994-01-01

    Second-site revertants from replication-incompetent molecular clones of human immunodeficiency virus (HIV) contain base substitutions adjacent to the TATA motif. The altered TATA box motifs were analyzed for their effect(s) on virus infectivity, long terminal repeat (LTR)-directed expression in transient transfection assays, in vitro RNA synthesis, and assembly of the TFIID-TFIIA preinitiation complex. The revertant TATA boxes accelerated the kinetics of HIV replication when present in the context of an LTR containing a Sp1 mutation (deletion or site specific); no effect was observed on the infectivity of wild-type HIV. In chloramphenicol acetyltransferase assays and in vitro transcription systems, the altered TATA box motifs led to elevated basal levels of RNA synthesis from NF-kappa B- and Sp1-mutagenized and wild-type templates, respectively, but did not increase responsiveness to Tat transactivation. The revertant TATA boxes accelerated the binding of TFIID and TFIIA to the LTR and stabilized their association with the promoter. The revertants did not assemble a more-processive elongation complex. These results suggest that in the context of an impaired enhancer/promoter (viz., three mutated Sp1 elements), a series of HIV revertants emerge which contain LTR alterations that significantly augment basal RNA synthesis. The TATA motif revertants are capable of rescuing the enhancer/promoter defect and sustain virus infectivity. Images PMID:8151790

  4. Science Prop Boxes

    ERIC Educational Resources Information Center

    Hommerding, Molly

    2007-01-01

    A prop box is a teacher-created resource full of age-appropriate and meaningful activities focused on a central theme. Prop boxes work much like learning centers in an elementary classroom with the important addition of providing opportunities for socio-dramatic play. Prop box play engages students in self-chosen activities that promote critical…

  5. Metagenomic insights into important microbes from the Dead Zone

    NASA Astrophysics Data System (ADS)

    Thrash, C.; Baker, B.; Seitz, K.; Temperton, B.; Gillies, L.; Rabalais, N. N.; Mason, O. U.

    2015-12-01

    Coastal regions of eutrophication-driven oxygen depletion are widespread and increasing in number. Also known as dead zones, these regions take their name from the deleterious effects of hypoxia (dissolved oxygen less than 2 mg/L) on shrimp, demersal fish, and other animal life. Dead zones result from nutrient enrichment of primary production, concomitant consumption by chemoorganotrophic aerobic microorganisms, and strong stratification that prevents ventilation of bottom water. One of the largest dead zones in the world occurs seasonally in the northern Gulf of Mexico (nGOM), where hypoxia can reach up to 22,000 square kilometers. While this dead zone shares many features with more well-known marine oxygen minimum zones, it is nevertheless understudied with regards to the microbial assemblages involved in biogeochemical cycling. We performed metagenomic and metatranscriptomic sequencing on six samples from the 2013 nGOM dead zone from both hypoxic and oxic bottom waters. Assembly and binning led to the recovery of over fifty partial to nearly complete metagenomes from key microbial taxa previously determined to be numerically abundant from 16S rRNA data, such as Thaumarcheaota, Marine Group II Euryarchaeota, SAR406, SAR324, Synechococcus spp., and Planctomycetes. These results provide information about the roles of these taxa in the nGOM dead zone, and opportunities for comparing this region of low oxygen to others around the globe.

  6. RNA helicase Belle/DDX3 regulates transgene expression in Drosophila.

    PubMed

    Lo, Pang-Kuo; Huang, Yi-Chun; Poulton, John S; Leake, Nicholas; Palmer, William H; Vera, Daniel; Xie, Gengqiang; Klusza, Stephen; Deng, Wu-Min

    2016-04-01

    Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we show that genetic inhibition of Bel function leads to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited. PMID:26900887

  7. Is Piaget's Epistemic Subject Dead?

    ERIC Educational Resources Information Center

    Lawson, Anton E.

    1991-01-01

    Argues that the Piaget's epistemic subject is not supported by evidence and contains weaknesses. Concludes that the epistemic subject is dead and that continued acceptance of this aspect of Piagetian theory would be counterproductive. (PR)

  8. Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3(+) regulatory T cells specific for the ubiquitous RNA-binding nuclear antigen La/Sjögren's syndrome (SS)-B.

    PubMed

    Pan, Z-J; Horton, C G; Lawrence, C; Farris, A D

    2016-10-01

    RNA-binding nuclear antigens are a major class of self-antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)-B (La), is controlled by CD4(+) T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa-specific CD4(+) T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte-replete recipient mice expressing hLa as a neo-self-antigen. After initial antigen-specific cell division, hLa-specific donor CD4(+) T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)-10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3-negative donor cells demonstrated that accumulation of hLa-specific regulatory T cells (Treg ) was due primarily to expansion of small numbers of donor Treg . Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3(+) donor Treg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co-stimulatory and co-inhibitory molecules than B cells. Adoptive transfer of hLa peptide-loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa-specific Treg . Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa-specific T cells impaired FoxP3(+) donor T cell accumulation. Therefore, peripheral expansion of Treg specific for an RNA-binding nuclear antigen is mediated by antigen-presenting pDC in a type 1 IFN-dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA-binding nuclear antigens.

  9. Climate change and dead zones.

    PubMed

    Altieri, Andrew H; Gedan, Keryn B

    2015-04-01

    Estuaries and coastal seas provide valuable ecosystem services but are particularly vulnerable to the co-occurring threats of climate change and oxygen-depleted dead zones. We analyzed the severity of climate change predicted for existing dead zones, and found that 94% of dead zones are in regions that will experience at least a 2 °C temperature increase by the end of the century. We then reviewed how climate change will exacerbate hypoxic conditions through oceanographic, ecological, and physiological processes. We found evidence that suggests numerous climate variables including temperature, ocean acidification, sea-level rise, precipitation, wind, and storm patterns will affect dead zones, and that each of those factors has the potential to act through multiple pathways on both oxygen availability and ecological responses to hypoxia. Given the variety and strength of the mechanisms by which climate change exacerbates hypoxia, and the rates at which climate is changing, we posit that climate change variables are contributing to the dead zone epidemic by acting synergistically with one another and with recognized anthropogenic triggers of hypoxia including eutrophication. This suggests that a multidisciplinary, integrated approach that considers the full range of climate variables is needed to track and potentially reverse the spread of dead zones.

  10. Climate change and dead zones.

    PubMed

    Altieri, Andrew H; Gedan, Keryn B

    2015-04-01

    Estuaries and coastal seas provide valuable ecosystem services but are particularly vulnerable to the co-occurring threats of climate change and oxygen-depleted dead zones. We analyzed the severity of climate change predicted for existing dead zones, and found that 94% of dead zones are in regions that will experience at least a 2 °C temperature increase by the end of the century. We then reviewed how climate change will exacerbate hypoxic conditions through oceanographic, ecological, and physiological processes. We found evidence that suggests numerous climate variables including temperature, ocean acidification, sea-level rise, precipitation, wind, and storm patterns will affect dead zones, and that each of those factors has the potential to act through multiple pathways on both oxygen availability and ecological responses to hypoxia. Given the variety and strength of the mechanisms by which climate change exacerbates hypoxia, and the rates at which climate is changing, we posit that climate change variables are contributing to the dead zone epidemic by acting synergistically with one another and with recognized anthropogenic triggers of hypoxia including eutrophication. This suggests that a multidisciplinary, integrated approach that considers the full range of climate variables is needed to track and potentially reverse the spread of dead zones. PMID:25385668

  11. Dead Star Rumbles

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site] Composite of Supernova Remnant Cassiopeia A This Spitzer Space Telescope composite shows the supernova remnant Cassiopeia A (white ball) and surrounding clouds of dust (gray, orange and blue). It consists of two processed images taken one year apart. Dust features that have not changed over time appear gray, while those that have changed are colored blue or orange. Blue represents an earlier time and orange, a later time.

    These observations illustrate that a blast of light from Cassiopeia A is waltzing outward through the dusty skies. This dance, called an 'infrared echo,' began when the remnant erupted about 50 years ago.

    Cassiopeia A is the remnant of a once massive star that died in a violent supernova explosion 325 years ago. It consists of a dead star, called a neutron star, and a surrounding shell of material that was blasted off as the star died. This remnant is located 10,000 light-years away in the northern constellation Cassiopeia.

    An infrared echo is created when a star explodes or erupts, flashing light into surrounding clumps of dust. As the light zips through the dust clumps, it heats them up, causing them to glow successively in infrared, like a chain of Christmas bulbs lighting up one by one. The result is an optical illusion, in which the dust appears to be flying outward at the speed of light. This apparent motion can be seen here by the shift in colored dust clumps.

    Echoes are distinct from supernova shockwaves, which are made up material that is swept up and hurled outward by exploding stars.

    This infrared echo is the largest ever seen, stretching more than 50 light-years away from Cassiopeia A. If viewed from Earth, the entire movie frame would take up the same amount of space as two full moons.

    Hints of an older infrared echo from Cassiopeia A's supernova explosion hundreds of years ago can also be seen.

    The earlier Spitzer image was taken on November 30

  12. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    PubMed

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans. PMID:26195740

  13. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    PubMed

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans.

  14. The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection.

    PubMed

    Wisskirchen, Christian; Ludersdorfer, Thomas H; Müller, Dominik A; Moritz, Eva; Pavlovic, Jovan

    2011-09-01

    The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

  15. An Improved Box Theater

    NASA Astrophysics Data System (ADS)

    Huster, Michael E.

    2011-09-01

    While designing an optics lab for a conceptual physics course, I came across a "box theater" activity. The box theater is a pinhole camera obscura made from a box that students put over their heads and shoulders. I use the activity as a capstone experience to explain optical systems. (Classroom demonstrations of the camera obscura have been described by others.2) First, the students build and experiment with a camera obscura made from a plastic cup and a convex lens with a focal length of 7.5 cm, and then "wear" the box theater. The difficulty with the box theater is the dimness of the image. A cloth drape has to be hung from the bottom of the box around the shoulders of the students to prevent light leakage, and the students have to wait a few minutes for their eyes to adjust to the darkness.

  16. Localization of Components of the RNA-Degrading Machine in Bacillus subtilis

    PubMed Central

    Cascante-Estepa, Nora; Gunka, Katrin; Stülke, Jörg

    2016-01-01

    In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient. PMID:27708634

  17. GLOVE BOX ATTACHMENT

    DOEpatents

    Butts, H.L.

    1962-02-13

    This invention comprises a housing unit to be fitted between a glove box port and a glove so that a slidable plate within the housing seals off the glove box port for evacuation of the glove box without damage to the glove. The housing and the glove may be evacuated without damage to the glove since movement of the glove is restricted during evacuation by the slidable plate. (AEC)

  18. [Parasitic dead-end: update].

    PubMed

    Magnaval, J F

    2006-08-01

    Parasitic dead-ends occur when a parasite is unable to establish a permanent interaction in an unnatural host. Although the likelihood of successful reproduction by the pathogenic agent is nul, parasitic dead-end heralds capture of new parasites and therefore expansion of the host range. Angiostrongyliasis due to A. cantonensis or A. costaricensis, anisakiasis, Ancylostoma caninum infection, gnathostomiasis and sparganosis are undoubtedly emerging zoonoses of particular medical interest. Prevention of these diseases relies on abstinence from eating raw meat from invertebrates or cold-blooded (poikilotherm) vertebrates (e.g. used in exotic dishes). These guidelines must be included in recommendations to travelers. PMID:16999036

  19. The mirror box

    NASA Astrophysics Data System (ADS)

    Thompson, Gene; Mathieson, Don

    2001-11-01

    The mirror box is an old standby in magic shows and an impressive demonstration of the law of reflection for the physics instructor. The box creates the illusion of an object floating in space by the use of a plane mirror.

  20. Thinking outside the Box

    ERIC Educational Resources Information Center

    Fanshawe, Simon; Sriskandarajah, Dhananjayan

    2010-01-01

    Britain is not only more diverse than ever before, but that diversity itself is growing more diverse. Britain's simplistic "tick-box" approach to identity is in danger of inhibiting the very equality it seeks to promote. To question the tick-box is not to accuse local authorities of "political correctness gone mad". The notion of political…

  1. Environmental Exchange Box

    ERIC Educational Resources Information Center

    Moseley, Christine

    2003-01-01

    In this activity, teachers in one state create and share an "exchange box" of environmental and cultural items with students of another state. The Environmental Exchange Box activity enables teachers to improve students' skills in scientific inquiry and develop attitudes and values conducive to science learning such as wonder, curiosity, and…

  2. Math in the Box

    ERIC Educational Resources Information Center

    DeYoung, Mary J.

    2009-01-01

    This article describes how to make an origami paper box and explores the algebra, geometry, and other mathematics that unfolds. A set of origami steps that transforms the paper into an open box can hold mathematical surprises for both students and teachers. An origami lesson can engage students in an open-ended exploration of the relationship…

  3. RNA chaperones buffer deleterious mutations in E. coli

    PubMed Central

    Rudan, Marina; Schneider, Dominique; Warnecke, Tobias; Krisko, Anita

    2015-01-01

    Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability. DOI: http://dx.doi.org/10.7554/eLife.04745.001 PMID:25806682

  4. Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae.

    PubMed

    Beck, Zachary T; Cloutier, Sara C; Schipma, Matthew J; Petell, Christopher J; Ma, Wai Kit; Tran, Elizabeth J

    2014-11-01

    Cellular homeostasis requires a fine balance between energy uptake, utilization, and growth. Dbp2 is a member of the DEAD-box protein family in Saccharomyces cerevisiae with characterized ATPase and helicase activity in vitro. DEAD-box RNA helicases are a class of enzymes that utilize ATP hydrolysis to remodel RNA and/or RNA-protein (RNP) composition. Dbp2 has been proposed to utilize its helicase activity in vivo to promote RNA-protein complex assembly of both messenger (m)RNAs and long noncoding (lnc)RNAs. Previous work from our laboratory demonstrated that loss of DBP2 enhances the lncRNA-dependent transcriptional induction of the GAL genes by abolishing glucose-dependent repression. Herein, we report that either a carbon source switch or glucose deprivation results in rapid export of Dbp2 to the cytoplasm. Genome-wide RNA sequencing identified a new class of antisense hexose transporter transcripts that are specifically upregulated upon loss of DBP2. Further investigation revealed that both sense and antisense hexose transporter (HXT) transcripts are aberrantly expressed in DBP2-deficient cells and that this expression pathway can be partially mimicked in wild-type cells by glucose depletion. We also find that Dbp2 promotes ribosome biogenesis and represses alternative ATP-producing pathways, as loss of DBP2 alters the transcript levels of ribosome biosynthesis (snoRNAs and associated proteins) and respiration gene products. This suggests that Dbp2 is a key integrator of nutritional status and gene expression programs required for energy homeostasis.

  5. Performance Evaluation: A Deadly Disease?

    ERIC Educational Resources Information Center

    Aluri, Rao; Reichel, Mary

    1994-01-01

    W. Edwards Deming condemned performance evaluations as a deadly disease afflicting American management. He argued that performance evaluations nourish fear, encourage short-term thinking, stifle teamwork, and are no better than lotteries. This article examines library literature from Deming's perspective. Although that literature accepts…

  6. Who are the Unclaimed Dead?

    PubMed

    Quinet, Kenna; Nunn, Samuel; Ballew, Alfarena

    2016-01-01

    Unclaimed dead are deceased persons with no known next of kin (NoK) or NoK was located but did not claim the deceased. Unclaimed dead in Marion County, Indiana, 2004-2011, are examined. Comparisons are provided of the unclaimed to the claimed dead population and county death patterns. Race, gender, marital status, age, location, manner and cause of death, NoK, and days to disposition are analyzed. The unclaimed dead were disproportionately male, slightly more likely to be Black, younger at death, died from natural causes, had unknown marital status, were equally likely as not to have NoK, did not die in a hospital, and were subject to autopsy. Nearly half the unclaimed had NoK who did not claim the body; the other half had no identifiable NoK. Unclaimed were more likely to have an autopsy and to die from external causes. Most unclaimed were identified by means outside fingerprints or DNA. PMID:26524620

  7. Structure and mechanism of the T-box riboswitches

    PubMed Central

    Zhang, Jinwei

    2015-01-01

    In most Gram-positive bacteria, including many clinically devastating pathogens from genera such as Bacillus, Clostridium, Listeria and Staphylococcus, T-box riboswitches sense and regulate intracellular availability of amino acids through a multipartite mRNA-tRNA interaction. The T-box mRNA leaders respond to nutrient starvation by specifically binding cognate tRNAs and sensing whether the bound tRNA is aminoacylated, as a proxy for amino acid availability. Based on this readout, T-boxes direct a transcriptional or translational switch to control the expression of downstream genes involved in various aspects of amino acid metabolism: biosynthesis, transport, aminoacylation, transamidation, etc. Two decades after its discovery, the structural and mechanistic underpinnings of the T-box riboswitch were recently elucidated, producing a wealth of insights into how two structured RNAs can recognize each other with robust affinity and exquisite selectivity. The T-box paradigm exemplifies how natural non-coding RNAs can interact not just through sequence complementarity, but can add molecular specificity by precisely juxtaposing RNA structural motifs, exploiting inherently flexible elements and the biophysical properties of post-transcriptional modifications, ultimately achieving a high degree of shape complementarity through mutually induced fit. The T-box also provides a proof-of-principle that compact RNA domains can recognize minute chemical changes (such as tRNA aminoacylation) on another RNA. The unveiling of the structure and mechanism of the T-box system thus expands our appreciation of the range of capabilities and modes of action of structured non-coding RNAs, and hints at the existence of networks of non-coding RNAs that communicate through both, structural and sequence specificity. PMID:25959893

  8. Glove box shield

    DOEpatents

    Brackenbush, L.W.; Hoenes, G.R.

    A shield for a glove box housing radioactive material is comprised of spaced apart clamping members which maintain three overlapping flaps in place therebetween. There is a central flap and two side flaps, the side flaps overlapping at the interior edges thereof and the central flap extending past the intersection of the side flaps in order to insure that the shield is always closed when the user wthdraws his hand from the glove box. Lead loaded neoprene rubber is the preferred material for the three flaps, the extent of lead loading depending upon the radiation levels within the glove box.

  9. Glove box shield

    DOEpatents

    Brackenbush, Larry W.; Hoenes, Glenn R.

    1981-01-01

    According to the present invention, a shield for a glove box housing radioactive material is comprised of spaced apart clamping members which maintain three overlapping flaps in place therebetween. There is a central flap and two side flaps, the side flaps overlapping at the interior edges thereof and the central flap extending past the intersection of the side flaps in order to insure that the shield is always closed when the user withdraws his hand from the glove box. Lead loaded neoprene rubber is the preferred material for the three flaps, the extent of lead loading depending upon the radiation levels within the glove box.

  10. The C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus aureus

    PubMed Central

    Giraud, Caroline; Hausmann, Stéphane; Lemeille, Sylvain; Prados, Julien; Redder, Peter; Linder, Patrick

    2015-01-01

    Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes. PMID:25997461

  11. FAQ: West Nile Virus and Dead Birds

    MedlinePlus

    ... Education Public Service Videos West Nile Virus & Dead Birds Recommend on Facebook Tweet Share Compartir On this ... dead bird sightings to local authorities. How do birds get infected with West Nile virus? West Nile ...

  12. Climate in a Box

    NASA Video Gallery

    NASA's Climate in a Box Project is exploring the utility of supercomputers in providing a complete, pre-packaged, ready-to-use toolkit of climate research products and on-demand access to a high-pe...

  13. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 5 2013-01-01 2013-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  14. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 5 2014-01-01 2014-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  15. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 5 2012-01-01 2012-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  16. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 5 2011-01-01 2011-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  17. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  18. Automatic box loader

    DOEpatents

    Eldridge, Harry H.; Jones, Robert A.; Lindner, Gordon M.; Hight, Paul H.

    1976-01-01

    This invention relates to a system for repetitively forming an assembly consisting of a single layer of tubes and a row of ferromagnetic armatures underlying the same, electromagnetically conveying the resulting assembly to a position overlying a storage box, and depositing the assembly in the box. The system includes means for simultaneously depositing a row of the armatures on the inclined surface of a tube retainer. Tubes then are rolled down the surface to form a single tube layer bridging the armatures. A magnet assembly carrying electromagnets respectively aligned with the armatures is advanced close to the tube layer, and in the course of this advance is angularly displaced to bring the pole pieces of the electromagnets into parallelism with the tube layer. The magnets then are energized to pick up the assembly. The loaded magnet assembly is retracted to a position overlying the box, and during this retraction is again displaced to bring the pole pieces of the electromagnets into a horizontal plane. Means are provided for inserting the loaded electromagnets in the box and then de-energizing the electromagnets to deposit the assembly therein. The system accomplishes the boxing of fragile tubes at relatively high rates. Because the tubes are boxed as separated uniform layers, subsequent unloading operations are facilitated.

  19. Normative seeds for deadly martyrdoms.

    PubMed

    Tobeña, Adolf; Vilarroya, Oscar

    2014-08-01

    Even if Lankford's biographical examination of perpetrators of suicidal attacks serves to alert us on the role played by individual factors in their recruitment, psychological frailties, distress, or coercion do not exhaust the causal pathways to deadly martyrdom. Normative personality attributes must be explored further in order to ascertain plausible roots of murderous sacrifice. We have advanced (Tobeña 2004b; 2009; 2011) a template of normative temperamental traits that could lead activists to the threshold of volunteering for murderous missions.

  20. Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation

    PubMed Central

    Weston, Andrew; Sommerville, John

    2006-01-01

    The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2α, bind to nascent transcripts on chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression, mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation, storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages of their lifetimes marks them out as key regulators of post-transcriptional gene expression. PMID:16769775

  1. Cable Tester Box

    NASA Technical Reports Server (NTRS)

    Lee, Jason H.

    2011-01-01

    Cables are very important electrical devices that carry power and signals across multiple instruments. Any fault in a cable can easily result in a catastrophic outcome. Therefore, verifying that all cables are built to spec is a very important part of Electrical Integration Procedures. Currently, there are two methods used in lab for verifying cable connectivity. (1) Using a Break-Out Box and an ohmmeter this method is time-consuming but effective for custom cables and (2) Commercial Automated Cable Tester Boxes this method is fast, but to test custom cables often requires pre-programmed configuration files, and cables used on spacecraft are often uniquely designed for specific purposes. The idea is to develop a semi-automatic continuity tester that reduces human effort in cable testing, speeds up the electrical integration process, and ensures system safety. The JPL-Cable Tester Box is developed to check every single possible electrical connection in a cable in parallel. This system indicates connectivity through LED (light emitting diode) circuits. Users can choose to test any pin/shell (test node) with a single push of a button, and any other nodes that are shorted to the test node, even if they are in the same connector, will light up with the test node. The JPL-Cable Tester Boxes offers the following advantages: 1. Easy to use: The architecture is simple enough that it only takes 5 minutes for anyone to learn how operate the Cable Tester Box. No pre-programming and calibration are required, since this box only checks continuity. 2. Fast: The cable tester box checks all the possible electrical connections in parallel at a push of a button. If a cable normally takes half an hour to test, using the Cable Tester Box will improve the speed to as little as 60 seconds to complete. 3. Versatile: Multiple cable tester boxes can be used together. As long as all the boxes share the same electrical potential, any number of connectors can be tested together.

  2. Box truss antenna technology status

    NASA Technical Reports Server (NTRS)

    Coyner, J. V.; Bachtell, E. E.

    1987-01-01

    Recent technology development activities for box truss structures and box truss antennas are summarized. Three primary activities are discussed: the development of an integrated analysis system for box truss mesh antennae; dynamic testing to characterize the effect of joint free play on the dynamic behavior of box truss structures; and fabrication of a 4.5 meter diameter offset fed mesh reflector integrated to an all graphite epoxy box truss cube.

  3. DDX1 is an RNA-dependent ATPase Involved in HIV-1 Rev Function and Virus Replication

    PubMed Central

    Edgcomb, Stephen P.; Carmel, Andrew B.; Naji, Souad; Ambrus-Aikelin, Geza; Reyes, Jason R.; Saphire, Andrew C.S.; Gerace, Larry; Williamson, James R.

    2011-01-01

    The HIV-1 Rev protein is essential for the virus because it promotes nuclear export of alternatively-processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in E. coli is a well-behaved folded protein. Binding assays using fluorescently-labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell based assays of HIV-1 production, and provide the first demonstration that recombinant DDX1 binds Rev and RNA, and has RNA dependent catalytic activity. PMID:22051512

  4. Are Brain Dead Individuals Dead? Grounds for Reasonable Doubt.

    PubMed

    Brugger, E Christian

    2016-06-01

    According to the biological definition of death, a human body that has not lost the capacity to holistically organize itself is the body of a living human individual. Reasonable doubt against the conclusion that it has lost the capacity exists when the body appears to express it and no evidence to the contrary is sufficient to rule out reasonable doubt against the conclusion that the apparent expression is a true expression (i.e., when the conclusion that what appears to be holistic organization is in fact holistic organization remains a reasonable explanatory hypothesis in light of the best evidence to the contrary). This essay argues that the evidence and arguments against the conclusion that the signs of complex bodily integration exhibited in ventilated brain dead bodies are true expressions of somatic integration are unpersuasive; that is, they are not adequate to exclude reasonable doubt against the conclusion that BD bodies are dead. Since we should not treat as corpses what for all we know might be living human beings, it follows that we have an obligation to treat BD individuals as if they were living human beings. PMID:27075192

  5. Depth in box spaces.

    PubMed

    Pont, Sylvia C; Nefs, Harold T; van Doorn, Andrea J; Wijntjes, Maarten W A; Te Pas, Susan F; de Ridder, Huib; Koenderink, Jan J

    2012-01-01

    Human observers adjust the frontal view of a wireframe box on a computer screen so as to look equally deep and wide, so that in the intended setting the box looks like a cube. Perspective cues are limited to the size-distance effect, since all angles are fixed. Both the size on the screen, and the viewing distance from the observer to the screen were varied. All observers prefer a template view of a cube over a veridical rendering, independent of picture size and viewing distance. If the rendering shows greater or lesser foreshortening than the template, the box appears like a long corridor or a shallow slab, that is, like a 'deformed' cube. Thus observers ignore 'veridicality'. This does not fit an 'inverse optics' model. We discuss a model of 'vision as optical user interface'.

  6. Is Piaget's epistemic subject dead?

    NASA Astrophysics Data System (ADS)

    Lawson, Anton E.

    Niaz (1990) presents arguments in favor of the retention of Piaget's epistemic subject as a theoretical construct to guide research and practice in science education and psychology. The intent of this article is to point out the weaknesses of those arguments and to suggest that the weight of evidence argues against the existence of the logical thinker postulated by Piaget. Therefore, contrary to Niaz's conclusion that the acceptance of Piaget's epistemic subject will facilitate the development of cognitive theories with greater explanatory power, the conclusion is reached that Piaget's epistemic subject is dead and that continued acceptance of this aspect of Piagetian theory would be counterproductive.

  7. "Dead quasars" in nearby galaxies?

    PubMed

    Rees, M J

    1990-02-16

    The nuclei of some galaxies undergo violent activity, quasars being the most extreme instances of this phenomenon. Such activity is probably short-lived compared to galactic lifetimes, and was most prevalent when the universe was only about one-fifth of its present age. A massive black hole seems the inevitable end point of such activity, and dead quasars should greatly outnumber active ones. In recent years, studies of stellar motions in the cores of several nearby galaxies indicate the presence of central dark masses which could be black holes. This article discusses how such evidence might be corroborated, and the potential implications for our understanding of active galaxies and black holes.

  8. Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

    PubMed Central

    Leitão, Ana Lúcia; Costa, Marina C.; Enguita, Francisco J.

    2015-01-01

    The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes. PMID:25622248

  9. The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing

    PubMed Central

    Zhang, Zhao; Wang, Jie; Schultz, Nadine; Zhang, Fan; Parhad, Swapnil S.; Tu, Shikui; Vreven, Thom; Zamore, Phillip D.; Weng, Zhiping; Theurkauf, William E.

    2014-01-01

    Summary piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino co-localizes to germline nuclear foci with Rai1/DXO related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced, and that rhino, cuff and uap56 mutations increase expression of spliced cluster transcripts over 100 fold. LacI∷Rhino fusion protein binding suppresses splicing of a reporter transgene, and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing, and speculate that stalled splicing differentiates piRNA precursors from mRNAs. PMID:24906152

  10. Unzippers, resolvers and sensors: a structural and functional biochemistry tale of RNA helicases.

    PubMed

    Leitão, Ana Lúcia; Costa, Marina C; Enguita, Francisco J

    2015-01-01

    The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

  11. Eating the dead in Madagascar.

    PubMed

    Campbell, Gwyn

    2013-12-01

    Cannibalism has been poorly understood and has seldom been studied, since it was often suppressed by missionaries and colonial administrators, and very few societies still practise it. Cannibalistic practices are more complex than was originally thought. They may be supported in societies under stress or in times of famine, to reflect aggression and antisocial behaviour (in cases where the bodies of enemies killed in battle or people who have harmed the family are eaten), or to honour a dead kinsman. It was, for example, noted in Madagascar during the imperial campaigns of Ranavalona I in the period 1829 - 1853. Two types of cannibalism have been described: exocannibalism, where enemies were consumed, and endocannibalism, where dead relatives were eaten to assist their passing to the world of the ancestors, or to prolong contact with beloved and admired family members and absorb their good qualities. This article reviews some of the beliefs and motivations that surrounded the cannibalistic practices of the people of Madagascar in the 19th century.  PMID:24300654

  12. Detection and quantification of RNA 2'-O-methylation and pseudouridylation.

    PubMed

    Huang, Chao; Karijolich, John; Yu, Yi-Tao

    2016-07-01

    RNA-guided RNA modification is a naturally occurring process that introduces 2'-O-methylation and pseudouridylation into rRNA, spliceosomal snRNA and several other types of RNA. The Box C/D ribonucleoproteins (RNP) and Box H/ACA RNP, each containing one unique guide RNA (Box C/D RNA or Box H/ACA RNA) and a set of core proteins, are responsible for 2'-O-methylation and pseudouridylation respectively. Box C/D RNA and Box H/ACA RNA provide the modification specificity through base pairing with their RNA substrate. These post-transcriptional modifications could profoundly alter the properties and functions of substrate RNAs. Thus it is desirable to establish reliable and standardized modification methods to study biological functions of modified nucleotides in RNAs. Here, we present several sensitive and efficient methods and protocols for detecting and quantifying post-transcriptional 2'-O-methylation and pseudouridylation. PMID:26853326

  13. 6. VIEW OF INTERIOR GLOVE BOX DURING CONSTRUCTION. GLOVE BOXES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. VIEW OF INTERIOR GLOVE BOX DURING CONSTRUCTION. GLOVE BOXES CONTAINED ALL PRODUCTION OPERATIONS AND WERE INTERCONNECTED BY CONVEYORS. (9/21/59) - Rocky Flats Plant, Plutonium Fabrication, Central section of Plant, Golden, Jefferson County, CO

  14. Looking Southwest at Reactor Box Furnaces With Reactor Boxes and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Looking Southwest at Reactor Box Furnaces With Reactor Boxes and Repossessed Uranium in Recycle Recovery Building - Hematite Fuel Fabrication Facility, Recycle Recovery Building, 3300 State Road P, Festus, Jefferson County, MO

  15. Teaching with Box Tops.

    ERIC Educational Resources Information Center

    Raiser, Lynne; D'Zamko, Mary Elizabeth

    1984-01-01

    Using environmental materials (such as the phone book and placemats from fast food restaurants) can be a motivating way to teach learning disabled students skills and concepts, as shown in an approach to reading, math, science and nutrition, and social studies instruction using a JELL-O brand gelatin box. (CL)

  16. Thinking "Inside" the Box

    ERIC Educational Resources Information Center

    Jeffries, Carolyn

    2011-01-01

    The authors conducted a test to determine whether they could incorporate a discovery box into a preschool setting was successful. It stimulated the students' natural inquiry processes while promoting understanding of healthy foods and allowing for practice of fine-motor skills. It was easily incorporated into the curriculum and classroom space.…

  17. Hydrophobic, Porous Battery Boxes

    NASA Technical Reports Server (NTRS)

    Bragg, Bobby J.; Casey, John E., Jr.

    1995-01-01

    Boxes made of porous, hydrophobic polymers developed to contain aqueous potassium hydroxide electrolyte solutions of zinc/air batteries while allowing air to diffuse in as needed for operation. Used on other types of batteries for in-cabin use in which electrolytes aqueous and from which gases generated during operation must be vented without allowing electrolytes to leak out.

  18. Cereal Box Totems.

    ERIC Educational Resources Information Center

    Jones, AnnMarie

    2002-01-01

    Presents a multicultural project used with fourth-grade students in which they created a three-dimensional totem pole using leftover cereal boxes. Discusses in detail how to create the totem pole. Explains that students learned about Northwest American Indians in class. (CMK)

  19. Sand-box modelling

    SciTech Connect

    Avery, P.

    1983-01-01

    As the result of an enquiry into BHRA's physical-reservoir-modelling experience, the use of sand box models was investigated. The type of model was considered a possible means of confirmation of a numerical model. The problem facing the numerical model user was comparing the performance of inclined or horizontal oil wells with that of the conventional vertical well.

  20. Science in a Box.

    ERIC Educational Resources Information Center

    Learning, 1991

    1991-01-01

    The ninth installment of the Science in a Box series (self-contained, hands-on activities for students grades three and up) presents activities for learning to use the sense of touch to determine how different objects conduct heat. All projects in the series are inexpensive and easy to assemble. (SM)

  1. Mystery Box Marvels

    ERIC Educational Resources Information Center

    Santos, Joel; Centurio, Tina

    2012-01-01

    What happens in the first week of school could very well set the stage for the rest of the school year. Setting high standards for science activities based in inquiry can start on the first day of science class and develop as the year unfolds. With the use of simple, readily available, inexpensive materials, an efficient mystery box lesson can be…

  2. "Can" the Black Box

    ERIC Educational Resources Information Center

    Lestingi, Francis S.

    1975-01-01

    Describes the use of the "Arcane (mysterious) Can" which is a "tin" can which is permanently sealed, both air- and water-tight, by means of a home canning device. The canning procedure permits the use of a large variety of materials which can not be utilized in the ordinary mystery box. This Can activity is valuable for illustrating in an…

  3. Shoe Box Circuits

    ERIC Educational Resources Information Center

    Sandifer, Cody

    2009-01-01

    Students' eyes grow wide with wonder as they get a motor to work or make a bulb light for the first time. As these daunting feats of electrical engineering remind us, teaching electricity is invariably rewarding and worthwhile. In this inquiry-based science project, elementary students work in pairs to design and wire a shoe box "room" that meets…

  4. "Dead quasars" in nearby galaxies?

    PubMed

    Rees, M J

    1990-02-16

    The nuclei of some galaxies undergo violent activity, quasars being the most extreme instances of this phenomenon. Such activity is probably short-lived compared to galactic lifetimes, and was most prevalent when the universe was only about one-fifth of its present age. A massive black hole seems the inevitable end point of such activity, and dead quasars should greatly outnumber active ones. In recent years, studies of stellar motions in the cores of several nearby galaxies indicate the presence of central dark masses which could be black holes. This article discusses how such evidence might be corroborated, and the potential implications for our understanding of active galaxies and black holes. PMID:17746076

  5. Hermit Points on a Box

    ERIC Educational Resources Information Center

    Hess, Richard; Grinstead, Charles; Grindstead, Marshall; Bergstrand, Deborah

    2008-01-01

    Suppose that we are given a rectangular box in 3-space. Given any two points on the surface of this box, we can define the surface distance between them to be the length of the shortest path between them on the surface of the box. This paper determines the pairs of points of maximum surface distance for all boxes. It is often the case that these…

  6. Making Connections with Memory Boxes.

    ERIC Educational Resources Information Center

    Whatley, April

    2000-01-01

    Addresses the use of children's literature within the social studies classroom on the topic of memory boxes. Includes discussions of four books: (1) "The Littlest Angel" (Charles Tazewell); (2) "The Hundred Penny Box" (Sharon Bell Mathis); (3) "Wilfrid Gordon McDonald Partridge" (Mem Fox); and (4) "The Memory Box" (Mary Bahr). (CMK)

  7. More Dead than Dead: Perceptions of Persons in the Persistent Vegetative State

    ERIC Educational Resources Information Center

    Gray, Kurt; Knickman, T. Anne; Wegner, Daniel M.

    2011-01-01

    Patients in persistent vegetative state (PVS) may be biologically alive, but these experiments indicate that people see PVS as a state curiously more dead than dead. Experiment 1 found that PVS patients were perceived to have less mental capacity than the dead. Experiment 2 explained this effect as an outgrowth of afterlife beliefs, and the…

  8. Box H/ACA small ribonucleoproteins.

    PubMed

    Kiss, Tamás; Fayet-Lebaron, Eléonore; Jády, Beáta E

    2010-03-12

    Box H/ACA RNAs represent an abundant, evolutionarily conserved class of small noncoding RNAs. All H/ACA RNAs associate with a common set of proteins, and they function as ribonucleoprotein (RNP) enzymes mainly in the site-specific pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Some H/ACA RNPs function in the nucleolytic processing of precursor rRNA (pre-rRNA) and synthesis of telomeric DNA. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. Recently, great progress has been made toward understanding of the biogenesis, intracellular trafficking, structure, and function of H/ACA RNPs.

  9. Molecular recognition of RhlB and RNase D in the Caulobacter crescentus RNA degradosome.

    PubMed

    Voss, Jarrod E; Luisi, Ben F; Hardwick, Steven W

    2014-12-01

    The endoribonuclease RNase E is a key enzyme in RNA metabolism for many bacterial species. In Escherichia coli, RNase E contributes to the majority of RNA turnover and processing events, and the enzyme has been extensively characterized as the central component of the RNA degradosome assembly. A similar RNA degradosome assembly has been described in the α-proteobacterium Caulobacter crescentus, with the interacting partners of RNase E identified as the Kreb's cycle enzyme aconitase, a DEAD-box RNA helicase RhlB and the exoribonuclease polynucleotide phosphorylase. Here we report that an additional degradosome component is the essential exoribonuclease RNase D, and its recognition site within RNase E is identified. We show that, unlike its E. coli counterpart, C. crescentus RhlB interacts directly with a segment of the N-terminal catalytic domain of RNase E. The crystal structure of a portion of C. crescentus RNase E encompassing the helicase-binding region is reported. This structure reveals that an inserted segment in the S1 domain adopts an α-helical conformation, despite being predicted to be natively unstructured. We discuss the implications of these findings for the organization and mechanisms of the RNA degradosome.

  10. Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

    PubMed Central

    Kaake, Robyn; Dawson, Anthony R.; Matheos, Dina; Nagashima, Kunio; Sitlani, Parth; Patterson, Kurt; Chang, Ivan; Huang, Lan; Sandmeyer, Suzanne

    2015-01-01

    Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs. PMID:26421679

  11. DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.

    PubMed

    Huang, Wendy; Thomas, Benjamin; Flynn, Ryan A; Gavzy, Samuel J; Wu, Lin; Kim, Sangwon V; Hall, Jason A; Miraldi, Emily R; Ng, Charles P; Rigo, Frank; Rigo, Frank W; Meadows, Sarah; Montoya, Nina R; Herrera, Natalia G; Domingos, Ana I; Rastinejad, Fraydoon; Myers, Richard M; Fuller-Pace, Frances V; Bonneau, Richard; Chang, Howard Y; Acuto, Oreste; Littman, Dan R

    2015-12-24

    T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases. PMID:26675721

  12. Torsion in box wings

    NASA Technical Reports Server (NTRS)

    Wheatley, John B

    1931-01-01

    Logical analysis of a box wing necessitates the allowance for the contribution of the drag spars to the torsional strength of the structure. A rigorous analysis is available in the use of the Method of Least Work. The best logical method of analysis is that applying Prandtl's Membrane Analogy. The results so obtained vary by a negligible amount from those obtained by the rigorous method.

  13. The Electronic Battle Box

    NASA Astrophysics Data System (ADS)

    Gouin, Denis; Turcotte, Guy; Lebel, Eric; Gilbert, Annie

    2000-08-01

    The Electronic Battle Box is an integrated suite of planning and decision-aid tools specially designed to facilitate Canadian Armed Force Officers during their training and during their tasks of preparing and conducting military operations. It is the result of a collaborative effort between the Defence Research Establishment Valcartier, the Directorate of Army Doctrine (DAD), the Directorate of Land Requirements (DLR), the G4 staff of 1Cdn Div HQ and CGI Information and Management Consultants Inc. Distributed on CD-ROM, the Electronic Battle Box contains efficient and user-friendly tools that significantly reduce the planning time for military operations and ensure staff officers a better focus on significant tasks. Among the tools are an OrBat Browser and an Equipment Browser allowing to view and edit military organizations, a Task Browser providing facilities to prepare plans using Gantt charts, a Logistic Planner allowing to estimate supply requirements applying complex calculations, and Road, Air and Rail Movement Planners. EBB also provides staff officers with a large set of doctrinal documents in an electronic format. This paper provides an overview of the various tools of the Electronic Battle Box.

  14. Respect for the dead and dying.

    PubMed

    Preus, A

    1984-11-01

    Against the thesis that permanently unconscious persons cannot be harmed, and thus are not owed moral deference, it is argued that even the dead can be harmed and are owed moral respect, so a fortiori those dubiously or not quite dead deserve some moral deference.

  15. There's Life in Those Dead Logs!

    ERIC Educational Resources Information Center

    Biggs, Devin; Miller, Todd; Hall, Dee

    2006-01-01

    Although it is unspectacular in appearance, dead wood is one of the most ecologically important resources in forests. Fallen logs, dead standing trees, stumps, and even cavities in live trees fulfill a wide range of roles. Prominent among these is that they provide habitat for many organisms, especially insects. Fourth-grade students at Fox…

  16. Profiles in garbage: Corrugated boxes

    SciTech Connect

    Miller, C.

    1997-12-01

    Corrugated boxes (also known as old corrugated containers, or OCC) are used to ship products to factories, warehouses, retail stores, offices, and homes. The primary market for OCC is the paperboard industry, which uses OCC for corrugated medium, linerboard, recycled paperboard, and other paper products. In addition, 2.6 million tons of OCC were exported in 1996. OCC provided 37% of the scrap paper that was exported in 1996. Some corrugated boxes can be reused before recycling. Corrugated boxes are easily and highly recyclable. Large producers such as grocery store warehouses and factories have recycled their corrugated boxes for some time. If shredded properly, uncoated corrugated boxes are easily compostable.

  17. Mortality due to infectious hematopoietic necrosis of sockeye salmon (Oncorhynchus nerka) fry in streamside egg incubation boxes

    USGS Publications Warehouse

    Mulcahy, D.; Pascho, R.J.; Jenes, C.K.

    1983-01-01

    Infectious hematopoietic necrosis virus caused mortality of sockeye salmon (Oncorhynchus nerka) in streamside egg incubation boxes. Virus was not detectable in eggs or alevins; its first isolation coincided with the appearance of dead fish in a trap on the outflow from the box. Mortality due to the virus did not occur in every egg box studied. However, when fry from the boxes were held in the laboratory, epizootics began as much as 3 wk later, with total mortality exceeding 90%. More than 96% of the dead fry had titers exceeding 105 plaque-forming units per gram. The peak incidence of virus in fry migrating in the river coincided with the arrival of hatchery-produced fry, although some fry believed to have been produced by natural spawning were also infected.Englis

  18. Small, Lightweight, Collapsible Glove Box

    NASA Technical Reports Server (NTRS)

    James, Jerry

    2009-01-01

    A small, lightweight, collapsible glove box enables its user to perform small experiments and other tasks. Originally intended for use aboard a space shuttle or the International Space Station (ISS), this glove box could also be attractive for use on Earth in settings in which work space or storage space is severely limited and, possibly, in which it is desirable to minimize weight. The development of this glove box was prompted by the findings that in the original space-shuttle or ISS setting, (1) it was necessary to perform small experiments in a large general-purpose work station, so that, in effect, they occupied excessive space; and it took excessive amounts of time to set up small experiments. The design of the glove box reflects the need to minimize the space occupied by experiments and the time needed to set up experiments, plus the requirement to limit the launch weight of the box and the space needed to store the box during transport into orbit. To prepare the glove box for use, the astronaut or other user has merely to insert hands through the two fabric glove ports in the side walls of the box and move two hinges to a locking vertical position (see figure). The user could do this while seated with the glove box on the user fs lap. When stowed, the glove box is flat and has approximately the thickness of two pieces of 8-in. (.20 cm) polycarbonate.

  19. Black box multigrid

    NASA Technical Reports Server (NTRS)

    Dendy, J. E., Jr.

    1981-01-01

    The black box multigrid (BOXMG) code, which only needs specification of the matrix problem for application in the multigrid method was investigated. It is contended that a major problem with the multigrid method is that each new grid configuration requires a major programming effort to develop a code that specifically handles that grid configuration. The SOR and ICCG methods only specify the matrix problem, no matter what the grid configuration. It is concluded that the BOXMG does everything else necessary to set up the auxiliary coarser problems to achieve a multigrid solution.

  20. The dead spot of a tennis racket

    NASA Astrophysics Data System (ADS)

    Cross, Rod

    1997-08-01

    It is shown that a tennis racket has a dead spot, but it does not have a well-defined sweet spot, when measured in terms of the rebound of a tennis ball. A ball dropped onto the center of the strings bounces to about 30% its original height. The bounce is much weaker near the tip of the racket, being almost zero at the dead spot. These effects are explained in terms of the effective mass and rotational inertia of the racket, and by reference to the behavior of other cantilevered beams. It is concluded, somewhat paradoxically, that the best place to hit a serve or smash is at the dead spot.

  1. Projection optics box

    DOEpatents

    Hale, Layton C.; Malsbury, Terry; Hudyma, Russell M.; Parker, John M.

    2000-01-01

    A projection optics box or assembly for use in an optical assembly, such as in an extreme ultraviolet lithography (EUVL) system using 10-14 nm soft x-ray photons. The projection optics box utilizes a plurality of highly reflective optics or mirrors, each mounted on a precision actuator, and which reflects an optical image, such as from a mask, in the EUVL system onto a point of use, such as a target or silicon wafer, the mask, for example, receiving an optical signal from a source assembly, such as a developed from laser system, via a series of highly reflective mirrors of the EUVL system. The plurality of highly reflective optics or mirrors are mounted in a housing assembly comprised of a series of bulkheads having wall members secured together to form a unit construction of maximum rigidity. Due to the precision actuators, the mirrors must be positioned precisely and remotely in tip, tilt, and piston (three degrees of freedom), while also providing exact constraint.

  2. DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

    PubMed Central

    Huang, Wendy; Thomas, Benjamin; Flynn, Ryan A.; Gavzy, Samuel J.; Wu, Lin; Kim, Sangwon V.; Hall, Jason A.; Miraldi, Emily R.; Ng, Charles P.; Rigo, Frank; Meadows, Sarah; Montoya, Nina R.; Herrera, Natalia G.; Domingos, Ana I.; Rastinejad, Fraydoon; Myers, Richard M.; Fuller-Pace, Frances V.; Bonneau, Richard; Chang, Howard Y.; Acuto, Oreste; Littman, Dan R.

    2016-01-01

    Th17 lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. We identified the DEAD-box RNA helicase DDX5 as a RORγt partner that coordinates transcription of selective Th17 genes and is required for Th17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and co-activate its targets depends on its intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in Cartilage-Hair Hypoplasia (CHH) patients. A targeted Rmrp mutation in mice, corresponding to one in CHH patients, abrogated the lncRNA’s chromatin recruitment, ability to potentiate DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation and promises new opportunities for therapeutic intervention in Th17-dependent diseases. PMID:26675721

  3. eIF4B, eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle.

    PubMed

    Harms, Ulf; Andreou, Alexandra Zoi; Gubaev, Airat; Klostermeier, Dagmar

    2014-07-01

    Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5'-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation. PMID:24848014

  4. Regular Exercise: Antidote for Deadly Diseases?

    MedlinePlus

    ... https://medlineplus.gov/news/fullstory_160326.html Regular Exercise: Antidote for Deadly Diseases? High levels of physical ... Aug. 9, 2016 (HealthDay News) -- Getting lots of exercise may reduce your risk for five common diseases, ...

  5. Surviving Sepsis: Taming a Deadly Immune Response

    MedlinePlus

    ... disclaimer . Subscribe Surviving Sepsis Taming a Deadly Immune Response Many people have never heard of sepsis, or ... tract infection) and then a powerful and harmful response by your body’s own immune system . “With sepsis, ...

  6. Dead pixel replacement in LWIR microgrid polarimeters.

    PubMed

    Ratliff, Bradley M; Tyo, J Scott; Boger, James K; Black, Wiley T; Bowers, David L; Fetrow, Matthew P

    2007-06-11

    LWIR imaging arrays are often affected by nonresponsive pixels, or "dead pixels." These dead pixels can severely degrade the quality of imagery and often have to be replaced before subsequent image processing and display of the imagery data. For LWIR arrays that are integrated with arrays of micropolarizers, the problem of dead pixels is amplified. Conventional dead pixel replacement (DPR) strategies cannot be employed since neighboring pixels are of different polarizations. In this paper we present two DPR schemes. The first is a modified nearest-neighbor replacement method. The second is a method based on redundancy in the polarization measurements.We find that the redundancy-based DPR scheme provides an order-of-magnitude better performance for typical LWIR polarimetric data. PMID:19547086

  7. The Classroom Animal: Box Turtles.

    ERIC Educational Resources Information Center

    Kramer, David C.

    1986-01-01

    Provides basic information on the anatomy, physiology, behaviors, and distribution patterns of the box turtle. Offers suggestions for the turtle's care and maintenance in a classroom environment. (ML)

  8. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Dead bodies. 71.55 Section 71.55 Public Health... QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable disease listed in § 71.32(b) may not be brought into a U.S. port unless the body is (a) properly embalmed...

  9. 42 CFR 71.55 - Dead bodies.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Dead bodies. 71.55 Section 71.55 Public Health... QUARANTINE Importations § 71.55 Dead bodies. The remains of a person who died of a communicable disease listed in § 71.32(b) may not be brought into a U.S. port unless the body is (a) properly embalmed...

  10. Dead zones and extrasolar planetary properties

    NASA Astrophysics Data System (ADS)

    Matsumura, Soko; Pudritz, Ralph E.

    2006-01-01

    Most low-mass protostellar discs evolve in clustered environments where they are affected by external radiation fields, while others evolve in more isolated star-forming regions. Assuming that the magnetorotational instability (MRI) is the main source of viscosity, we calculate the size of a poorly ionized, MRI inactive and hence low viscosity region - the `dead zone'- in these protostellar discs. We include disc ionization by X-rays, cosmic rays, radioactive elements and thermal collisions, recombination by molecules, metals and grains, as well as the effect of turbulence stimulation in the dead zone by the active layers lying above it. We also calculate the gap-opening masses of planets, which are determined by a disc's viscosity and a disc aspect ratio, for discs in these environments and compare them with each other. We find that the dead zone is a robust feature of the protostellar discs that is largely independent of their environment, typically stretching out to ~15 au. We analyse the possible effects of dead zones on planet formation, migration and eccentricity evolution. We show that the gap-opening mass inside the dead zone is expected to be of the order of terrestrial and ice giant mass planets while that outside the dead zone is Jovian or super-Jovian mass planets, largely independent of the star-forming environment. We show that dead zones can significantly slow down both type I and type II planetary migration due to their lower viscosity. We also find that the growth of eccentricity of massive extrasolar planets is particularly favourable through the planet-disc interaction inside the dead zones due to the large gaps expected to be opened by planets.

  11. 2. UPPER NOTTINGHAM MINE, WOODEN BOXES. BOXES ARE LOCATED APPROXIMATELY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. UPPER NOTTINGHAM MINE, WOODEN BOXES. BOXES ARE LOCATED APPROXIMATELY 10 YARDS TO THE RIGHT AND DOWNSLOPE OF THE ADIT IN ID-31-F-1. CAMERA IS POINTED EAST-SOUTHEAST. - Florida Mountain Mining Sites, Upper Nottingham Mine, West face of Florida Mountain, head of Jacobs Gulch, Silver City, Owyhee County, ID

  12. ACYSYS in a box

    SciTech Connect

    Briegel, C.; Finstrom, D.; Hendricks, B.; King, C.; Lackey, S.; Neswold, R.; Nicklaus, D.; Patrick, J.; Petrov, A.; Rechenmacher, R.; Schumann, C.; /Fermilab

    2011-11-01

    The Accelerator Control System at Fermilab has evolved to enable this relatively large control system to be encapsulated into a 'box' such as a laptop. The goal was to provide a platform isolated from the 'online' control system. This platform can be used internally for making major upgrades and modifications without impacting operations. It also provides a standalone environment for research and development including a turnkey control system for collaborators. Over time, the code base running on Scientific Linux has enabled all the salient features of the Fermilab's control system to be captured in an off-the-shelf laptop. The anticipated additional benefits of packaging the system include improved maintenance, reliability, documentation, and future enhancements.

  13. Impedance Measurement Box

    ScienceCinema

    Christophersen, Jon

    2016-07-12

    Energy storage devices, primarily batteries, are now more important to consumers, industries and the military. With increasing technical complexity and higher user expectations, there is also a demand for highly accurate state-of-health battery assessment techniques. IMB incorporates patented, proprietary, and tested capabilities using control software and hardware that can be part of an embedded monitoring system. IMB directly measures the wideband impedance spectrum in seconds during battery operation with no significant impact on service life. It also can be applied to batteries prior to installation, confirming health before entering active service, as well as during regular maintenance. For more information about this project, visit http://www.inl.gov/rd100/2011/impedance-measurement-box/

  14. Impedance Measurement Box

    SciTech Connect

    Christophersen, Jon

    2011-01-01

    Energy storage devices, primarily batteries, are now more important to consumers, industries and the military. With increasing technical complexity and higher user expectations, there is also a demand for highly accurate state-of-health battery assessment techniques. IMB incorporates patented, proprietary, and tested capabilities using control software and hardware that can be part of an embedded monitoring system. IMB directly measures the wideband impedance spectrum in seconds during battery operation with no significant impact on service life. It also can be applied to batteries prior to installation, confirming health before entering active service, as well as during regular maintenance. For more information about this project, visit http://www.inl.gov/rd100/2011/impedance-measurement-box/

  15. The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

    NASA Technical Reports Server (NTRS)

    Oren, A.; Lau, P. P.; Fox, G. E.

    1988-01-01

    A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.

  16. Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.

    PubMed

    Lai, Eric C; Tam, Bergin; Rubin, Gerald M

    2005-05-01

    Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.

  17. DEAD-box helicase DP103 defines metastatic potential of human breast cancers

    PubMed Central

    Shin, Eun Myoung; Sin Hay, Hui; Lee, Moon Hee; Goh, Jen Nee; Tan, Tuan Zea; Sen, Yin Ping; Lim, See Wee; Yousef, Einas M.; Ong, Hooi Tin; Thike, Aye Aye; Kong, Xiangjun; Wu, Zhengsheng; Mendoz, Earnest; Sun, Wei; Salto-Tellez, Manuel; Lim, Chwee Teck; Lobie, Peter E.; Lim, Yoon Pin; Yap, Celestial T.; Zeng, Qi; Sethi, Gautam; Lee, Martin B.; Tan, Patrick; Goh, Boon Cher; Miller, Lance D.; Thiery, Jean Paul; Zhu, Tao; Gaboury, Louis; Tan, Puay Hoon; Hui, Kam Man; Yip, George Wai-Cheong; Miyamoto, Shigeki; Kumar, Alan Prem; Tergaonkar, Vinay

    2014-01-01

    Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β–activated kinase-1 (TAK1) phosphorylation of NF-κB–activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB–mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment. PMID:25083991

  18. Cardboard Boxes: Learning Concepts Galore!

    ERIC Educational Resources Information Center

    Warner, Laverne; Wilmoth, Linda

    2007-01-01

    Mrs. Keenan, a preschool teacher, observed her 3-year-old granddaughter Riley pull, tug, and stack piles of holiday boxes on the floor. She remembered that her child care director had suggested using boxes as a curriculum theme, but she hadn't given much thought about the idea until now. She said to herself, "I wonder if my children would be as…

  19. What Makes a Better Box?

    ERIC Educational Resources Information Center

    Moyer, Richard; Everett, Susan

    2010-01-01

    Every morning, many Americans start their day with a bowl of cereal. Some spend time while they eat breakfast reading the back of the cereal box, but few consider its size, shape, and construction, or realize that it was designed by an engineer. This article describes a lesson in which students design, build, and critique cereal boxes. The lesson…

  20. Spirit Boxes: Expressions of Culture.

    ERIC Educational Resources Information Center

    DeMuro, Ted

    1984-01-01

    After studying the culture and art of the ancient civilizations of South America, Mesopotamia, Greece, and Egypt, secondary level art students made spirit boxes as expressions of the various cultures. How to make the boxes and how to prepare the face molds are described. (RM)

  1. Box-and-Whisker Plots.

    ERIC Educational Resources Information Center

    Larsen, Russell D.

    1985-01-01

    Box-and-whisker plots (which give rapid visualization of batches of data) can be effectively used to present diverse collections of data used in traditional first-year chemistry courses. Construction of box-and-whisker plots and their use with bond energy data and data on heats of formation and solution are discussed. (JN)

  2. Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication

    PubMed Central

    Li, Yinzi; Xiong, Ruyi; Bernards, Mark; Wang, Aiming

    2016-01-01

    Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb. PMID:27456972

  3. Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication.

    PubMed

    Li, Yinzi; Xiong, Ruyi; Bernards, Mark; Wang, Aiming

    2016-01-01

    Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb. PMID:27456972

  4. Can the dead be brought into disrepute?

    PubMed

    Masterton, Malin; Hansson, Mats G; Höglund, Anna T; Helgesson, Gert

    2007-01-01

    Queen Christina of Sweden was unconventional in her time, leading to hypotheses on her gender and possible hermaphroditic nature. If genetic analysis can substantiate the latter claim, could this bring the queen into disrepute 300 years after her death? Joan C. Callahan has argued that if a reputation changes, this constitutes a change only in the group of people changing their views and not in the person whose reputation it is. Is this so? This paper analyses what constitutes change and draws out the implications to the reputation of the dead. It is argued that a reputation is a relational property which can go through changes. The change is "real" for the group changing their views on Queen Christina and of a Cambridge kind for the long dead queen herself. Cambridge changes result in new properties being acquired, some of which can be of significance. Although the dead cannot go through any non-relational changes, it is possible for the dead to change properties through Cambridge changes. In this sense changes in reputation do affect the dead, and thus Queen Christina can acquire a new property, in this case possibly a worse reputation. PMID:17549606

  5. Conserved boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs.

    PubMed Central

    Lange, T S; Borovjagin, A; Maxwell, E S; Gerbi, S A

    1998-01-01

    Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates. PMID:9606199

  6. Thinking Inside the Box

    SciTech Connect

    Boeheim, Charles T.; /SLAC

    2007-11-16

    In early 2007, SLAC was faced with a shortage of both electrical power and cooling in the main computer building, at the same time that the BaBar collaboration needed a new cluster of 250 batch machines installed. A number of different options were explored for the expansion. Provision of additional electrical power to the building was estimated to take one to two years, and cost several million dollars; additional cooling was even worse. Space in a Silicon Valley co-location facilities was reasonable on a one-year timescale, but broke even in costs by the end of three years, and were more expensive after that. There were also unresolved questions about the affects of additional latency from an offsite compute cluster to the onsite disk servers. The option of converting existing experimental hall space into computer space was estimated at one year, with uncertain availability. An option to aggressively replace several existing clusters with more power-efficient equipment was studied closely, but was disruptive to continued operations, expensive, and didn't provide any additional headroom. Finally, the installation of a Sun Project Blackbox (PBB) unit was selected as providing the capacity on a timescale of six months for a reasonable cost with minimal disruption to service. SLAC obtained and installed a beta unit and have been running it in production since September 2007. The experiences described are with the Early Access version of the PBB. The production version of the box has engineering changes based in part on our experiences.

  7. Dead Zone Accretion Flows in Protostellar Disks

    NASA Technical Reports Server (NTRS)

    Turner, Neal; Sano, T.

    2008-01-01

    Planets form inside protostellar disks in a dead zone where the electrical resistivity of the gas is too high for magnetic forces to drive turbulence. We show that much of the dead zone nevertheless is active and flows toward the star while smooth, large-scale magnetic fields transfer the orbital angular momentum radially outward. Stellar X-ray and radionuclide ionization sustain a weak coupling of the dead zone gas to the magnetic fields, despite the rapid recombination of free charges on dust grains. Net radial magnetic fields are generated in the magnetorotational turbulence in the electrically conducting top and bottom surface layers of the disk, and reach the midplane by ohmic diffusion. A toroidal component to the fields is produced near the midplane by the orbital shear. The process is similar to the magnetization of the solar tachocline. The result is a laminar, magnetically driven accretion flow in the region where the planets form.

  8. Effect of dead material in a calorimeter

    SciTech Connect

    Green, D.

    1995-10-01

    The existence of dead material in any practical calorimeter system is simply a fact of life. The task for the designer, then, is to understand the impact on the Physics in question, and strive to minimize it. The aim of this note is to use the ``Hanging File`` test data, which has fined grained individual readout of about 100 depth segments, to explore impact of dead material on the mean and r.m.s. of the hadronic distribution. The amount and location of the dead material is varied. It important to remember that the Hanging File data was calibrated, EM to HCAL compartment, so as to minimize the electron to pion energy dependence. In practical terms e/pie was made = 1.0 at an incident energy of about 100 GeV. Note that the PB(EM) + FE(HCAL) calorimeter was not a compensating device.

  9. Microbial and Chemical Characterization of Underwater Fresh Water Springs in the Dead Sea

    PubMed Central

    Ionescu, Danny; Siebert, Christian; Polerecky, Lubos; Munwes, Yaniv Y.; Lott, Christian; Häusler, Stefan; Bižić-Ionescu, Mina; Quast, Christian; Peplies, Jörg; Glöckner, Frank Oliver; Ramette, Alban; Rödiger, Tino; Dittmar, Thorsten; Oren, Aharon; Geyer, Stefan; Stärk, Hans-Joachim; Sauter, Martin; Licha, Tobias; Laronne, Jonathan B.; de Beer, Dirk

    2012-01-01

    Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water’s chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea−Dead Sea water conduit. PMID:22679498

  10. Microbial and chemical characterization of underwater fresh water springs in the Dead Sea.

    PubMed

    Ionescu, Danny; Siebert, Christian; Polerecky, Lubos; Munwes, Yaniv Y; Lott, Christian; Häusler, Stefan; Bižić-Ionescu, Mina; Quast, Christian; Peplies, Jörg; Glöckner, Frank Oliver; Ramette, Alban; Rödiger, Tino; Dittmar, Thorsten; Oren, Aharon; Geyer, Stefan; Stärk, Hans-Joachim; Sauter, Martin; Licha, Tobias; Laronne, Jonathan B; de Beer, Dirk

    2012-01-01

    Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit. PMID:22679498

  11. Dead-time Corrected Disdrometer Data

    DOE Data Explorer

    Bartholomew, Mary Jane

    2008-03-05

    Original and dead-time corrected disdrometer results for observations made at SGP and TWP. The correction is based on the technique discussed in Sheppard and Joe, 1994. In addition, these files contain calculated radar reflectivity factor, mean Doppler velocity and attenuation for every measurement for both the original and dead-time corrected data at the following wavelengths: 0.316, 0.856, 3.2, 5, and 10cm (W,K,X,C,S bands). Pavlos Kollias provided the code to do these calculations.

  12. Box truss development and applications

    NASA Astrophysics Data System (ADS)

    Coyner, J. V., Jr.

    1983-05-01

    The development and applications of a box truss design for large space antennas are discussed. A kinematic model with a mesh reflector was constructed. A prototype cube is described. Details of fabrication are given.

  13. MODELING MAGNETOROTATIONAL TURBULENCE IN PROTOPLANETARY DISKS WITH DEAD ZONES

    SciTech Connect

    Okuzumi, Satoshi; Hirose, Shigenobu

    2011-12-01

    Turbulence driven by magnetorotational instability (MRI) crucially affects the evolution of solid bodies in protoplanetary disks. On the other hand, small dust particles stabilize MRI by capturing ionized gas particles needed for the coupling of the gas and magnetic fields. To provide an empirical basis for modeling the coevolution of dust and MRI, we perform three-dimensional, ohmic-resistive MHD simulations of a vertically stratified shearing box with an MRI-inactive 'dead zone' of various sizes and with a net vertical magnetic flux of various strengths. We find that the vertical structure of turbulence is well characterized by the vertical magnetic flux and three critical heights derived from the linear analysis of MRI in a stratified disk. In particular, the turbulent structure depends on the resistivity profile only through the critical heights and is insensitive to the details of the resistivity profile. We discover scaling relations between the amplitudes of various turbulent quantities (velocity dispersion, density fluctuation, vertical diffusion coefficient, and outflow mass flux) and vertically integrated accretion stresses. We also obtain empirical formulae for the integrated accretion stresses as a function of the vertical magnetic flux and the critical heights. These empirical relations allow us to predict the vertical turbulent structure of a protoplanetary disk for a given strength of the magnetic flux and a given resistivity profile.

  14. The lithium vapor box divertor

    NASA Astrophysics Data System (ADS)

    Goldston, R. J.; Myers, R.; Schwartz, J.

    2016-02-01

    It has long been recognized that volumetric dissipation of the plasma heat flux from a fusion power system is preferable to its localized impingement on a material surface. Volumetric dissipation mitigates both the anticipated very high heat flux and intense particle-induced damage due to sputtering. Recent projections to a tokamak demonstration power plant suggest an immense upstream parallel heat flux, of order 20 GW m-2, implying that fully detached operation may be a requirement for the success of fusion power. Building on pioneering work on the use of lithium by Nagayama et al and by Ono et al as well as earlier work on the gas box divertor by Watkins and Rebut, we present here a concept for a lithium vapor box divertor, in which lithium vapor extracts momentum and energy from a fusion-power-plant divertor plasma, using fully volumetric processes. At the high powers and pressures that are projected this requires a high density of lithium vapor, which must be isolated from the main plasma in order to avoid lithium build-up on the chamber walls or in the plasma. Isolation is achieved through a powerful multi-box differential pumping scheme available only for condensable vapors. The preliminary box-wise calculations are encouraging, but much more work is required to demonstrate the practical viability of this scheme, taking into account at least 2D plasma and vapor flows within and between the vapor boxes and out of the vapor boxes to the main plasma.

  15. Hydrocarbon potential of Dead Sea Rift valley

    SciTech Connect

    Wilson, J.E.; Kashai, E.L.; Croker, P.F.

    1983-03-01

    The Dead Sea Rift is one of the world's unique geologic and topographic features, whose petroleum potential has not yet been evaluated. The sector of the Dead Sea is an asymmetric graben 20 km (12 mi) from rim to rim and over 120 km (75 mi) long. The total throw from the west rim, where the Upper Cretaceous crops out to the deeper portion of the grabens, is more than 8 km (26,200 ft). Throw on the eastern side is considerably greater as the valley wall is largely Precambrian. The level of the Dead Sea is -400 m (-1300 ft) - the lowest place on earth. Asphalt blocks floating from the Dead Sea, along with asphalt and heavy oil seeps in the valley, have been known since biblical times. These are suggestive of leaks from deeper accumulations. Although some exploration drilling has been done, no test has yet reached objectives in the deeper sunken block where the Miocene is figured to be at a depth of at least 7 km (23,000 ft.)

  16. Unethical and Deadly Symbiosis in Higher Education

    ERIC Educational Resources Information Center

    Crumbley, D. Larry; Flinn, Ronald; Reichelt, Kenneth J.

    2012-01-01

    As administrators are pressured to increase retention rates in accounting departments, and higher education in general, a deadly symbiosis is occurring. Most students and parents only wish for high grades, so year after year many educators engage in unethical grade inflation and course work deflation. Since administrators use the students to audit…

  17. Cheatgrass Dead Zones in Northern Nevada

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reports of areas of cheatgrass die-off are becoming more frequent. In 2009, we investigated cheatgrass die-off in north-central Nevada. Dead zones ranged from several to hundreds of acres in size and were largely unvegetated and covered by cheatgrass litter with a distinct gray cast. We collected re...

  18. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... and standards for protection of nuclear weapons (paragraph (c) of this section AR 50-5-1) and for... weapon). Use it only in extreme need, when all lesser means have failed or cannot reasonably be used. Use... property (such as operable weapons or ammunition) which could cause deadly harm to others in the hands...

  19. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... and standards for protection of nuclear weapons (paragraph (c) of this section AR 50-5-1) and for... weapon). Use it only in extreme need, when all lesser means have failed or cannot reasonably be used. Use... property (such as operable weapons or ammunition) which could cause deadly harm to others in the hands...

  20. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... and standards for protection of nuclear weapons (paragraph (c) of this section AR 50-5-1) and for... weapon). Use it only in extreme need, when all lesser means have failed or cannot reasonably be used. Use... property (such as operable weapons or ammunition) which could cause deadly harm to others in the hands...

  1. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... and standards for protection of nuclear weapons (paragraph (c) of this section AR 50-5-1) and for... weapon). Use it only in extreme need, when all lesser means have failed or cannot reasonably be used. Use... property (such as operable weapons or ammunition) which could cause deadly harm to others in the hands...

  2. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... and standards for protection of nuclear weapons (paragraph (c) of this section AR 50-5-1) and for... weapon). Use it only in extreme need, when all lesser means have failed or cannot reasonably be used. Use... property (such as operable weapons or ammunition) which could cause deadly harm to others in the hands...

  3. Effect of Dead Algae on Soil Permeability

    SciTech Connect

    Harvey, R.S.

    2003-02-21

    Since existing basins support heavy growths of unicellular green algae which may be killed by temperature variation or by inadvertent pH changes in waste and then deposited on the basin floor, information on the effects of dead algae on soil permeability was needed. This study was designed to show the effects of successive algal kills on the permeability of laboratory soil columns.

  4. Membrane Recognition and Dynamics of the RNA Degradosome

    PubMed Central

    Strahl, Henrik; Turlan, Catherine; Khalid, Syma; Bond, Peter J.; Kebalo, Jean-Marie; Peyron, Pascale; Poljak, Leonora; Bouvier, Marie; Hamoen, Leendert; Luisi, Ben F.; Carpousis, Agamemnon J.

    2015-01-01

    RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies. PMID:25647427

  5. 30 CFR 57.12006 - Distribution boxes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Distribution boxes. 57.12006 Section 57.12006... and Underground § 57.12006 Distribution boxes. Distribution boxes shall be provided with a... deenergized, and the distribution box shall be labeled to show which circuit each device controls....

  6. 30 CFR 57.12006 - Distribution boxes.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Distribution boxes. 57.12006 Section 57.12006... and Underground § 57.12006 Distribution boxes. Distribution boxes shall be provided with a... deenergized, and the distribution box shall be labeled to show which circuit each device controls....

  7. 30 CFR 57.12006 - Distribution boxes.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Distribution boxes. 57.12006 Section 57.12006... and Underground § 57.12006 Distribution boxes. Distribution boxes shall be provided with a... deenergized, and the distribution box shall be labeled to show which circuit each device controls....

  8. Plate forming and break down pizza box

    DOEpatents

    Pantisano, Frank; Devine, Scott M.

    1992-01-01

    A standard corrugated paper pizza box is provided with slit cuts cut through the top panel of the pizza box in a shape to form four circular serving plates with a beveled raised edge and cross slit cuts through the bottom panel of the pizza box separating the box into four essentially equal portions for easy disposal.

  9. Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes.

    PubMed Central

    Ladomery, M; Wade, E; Sommerville, J

    1997-01-01

    In investigating the composition of stored (maternal) mRNP particles in Xenopus oocytes, attention has focussed primarily on the phosphoproteins pp60/56, which are Y-box proteins involved in a general packaging of mRNA. We now identify a third, abundant, integral component of stored mRNP particles, Xp54, which belongs to the family of DEAD-box RNA helicases. Xp54 was first detected by its ability to photocrosslink ATP. Subsequent sequence analysis identifies Xp54 as a member of a helicase subfamily which includes: human p54, encoded at a chromosomal breakpoint in the B-cell lymphoma cell line, RC-K8; Drosophila ME31B, encoded by a maternally-expressed gene, and Saccharomyces pombe Ste13, cloned by complementation of the sterility mutant ste13. Expression studies reveal that the gene encoding Xp54 is transcribed maximally at early oogenesis: no transcripts are detected in adult tissues, other than ovary. Using a monospecific antibody raised against native Xp54, its presence in mRNP particles is confirmed by immunoblotting fractions bound to oligo(dT)-cellulose and separated by rate sedimentation and buoyant density. On isolating Xp54 from mRNP particles, it is shown to possess an ATP-dependent RNA helicase activity. Possible functions of Xp54 are discussed in relation to the assembly and utilization of mRNP particles. PMID:9023105

  10. More dead than dead: perceptions of persons in the persistent vegetative state.

    PubMed

    Gray, Kurt; Knickman, T Anne; Wegner, Daniel M

    2011-11-01

    Patients in persistent vegetative state (PVS) may be biologically alive, but these experiments indicate that people see PVS as a state curiously more dead than dead. Experiment 1 found that PVS patients were perceived to have less mental capacity than the dead. Experiment 2 explained this effect as an outgrowth of afterlife beliefs, and the tendency to focus on the bodies of PVS patients at the expense of their minds. Experiment 3 found that PVS is also perceived as "worse" than death: people deem early death better than being in PVS. These studies suggest that people perceive the minds of PVS patients as less valuable than those of the dead - ironically, this effect is especially robust for those high in religiosity. PMID:21798528

  11. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

    PubMed Central

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

  12. Chronic Infections of West Nile Virus Detected in California Dead Birds

    PubMed Central

    Padgett, Kerry; Fang, Ying; Woods, Leslie; Foss, Leslie; Anderson, Jaynia; Kramer, Vicki

    2013-01-01

    Abstract During 2010 and 2011, 933 recently deceased birds, submitted as part of the dead bird surveillance program, tested positive for West Nile virus RNA at necropsy. The relative amount of RNA measured by qRT-PCR cycles ranged from 8.2 to 37.0 cycle threshold (Ct) and formed a bimodal frequency distribution, with maxima at 20 and 36 Ct and minima at 28–30 Ct. On the basis of frequency distributions among different avian species with different responses to infection following experimental inoculation, field serological data indicating survival of infection, and the discovery of persistent RNA in experimentally infected birds, dead birds collected in nature were scored as “recent” or “chronic” infections on the basis of Ct scores. The percentage of birds scored as having chronic infections was highest during late winter/spring, when all birds were after hatching year, and lowest during late summer, when enzootic transmission was typically highest as indicated by mosquito infections. Our data indicated that intervention efforts should not be based on dead birds with chronic infections unless supported by additional surveillance metrics. PMID:23488452

  13. period-1 encodes an ATP-dependent RNA helicase that influences nutritional compensation of the Neurospora circadian clock

    PubMed Central

    Emerson, Jillian M.; Bartholomai, Bradley M.; Ringelberg, Carol S.; Baker, Scott E.; Loros, Jennifer J.; Dunlap, Jay C.

    2015-01-01

    Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 [DEAD (Asp-Glu-Ala-Asp) Box Helicase 5] and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. Although prd-1 mutants display a long period (∼25 h) circadian developmental cycle, they interestingly display a WT period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator in the prd-1 mutant strain runs with a long period under glucose-sufficient conditions. Thus, PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein, and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose, PRD-1 is in the nucleus until glucose runs out, which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd-1 may be formally viewed as a clock mutant with defective nutritional compensation of circadian period length. PMID:26647184

  14. Gulf of Mexico dead zone - the last 150 years

    USGS Publications Warehouse

    Osterman, Lisa; Swarzenski, P.W.; Poore, R.Z.

    2006-01-01

    'Gulf of Mexico Dead Zone-The Last 150 Years' discusses the dead zone that forms seasonally in the northern Gulf of Mexico when subsurface waters become depleted in dissolved oxygen and cannot support most life.

  15. TATA box polymorphisms in human gene promoters and associated hereditary pathologies.

    PubMed

    Savinkova, L K; Ponomarenko, M P; Ponomarenko, P M; Drachkova, I A; Lysova, M V; Arshinova, T V; Kolchanov, N A

    2009-02-01

    TATA-binding protein (TBP) is the first basal factor that recognizes and binds a TATA box on TATA-containing gene promoters transcribed by RNA polymerase II. Data available in the literature are indicative of admissible variability of the TATA box. The TATA box flanking sequences can influence TBP affinity as well as the level of basal and activated transcription. The possibility of mediated involvement in in vivo gene expression regulation of the TBP interactions with variant TATA boxes is supported by data on TATA box polymorphisms and associated human hereditary pathologies. A table containing data on TATA element polymorphisms in human gene promoters (about 40 mutations have been described), associated with particular pathologies, their short functional characteristics, and manifestation mechanisms of TATA-box SNPs is presented. Four classes of polymorphisms are considered: TATA box polymorphisms that weaken and enhance promoter, polymorphisms causing TATA box emergence and disappearance, and human virus TATA box polymorphisms. The described examples are indicative of the polymorphism-associated severe pathologies like thalassemia, the increased risk of hepatocellular carcinoma, sensitivity to H. pylori infection, oral cavity and lung cancers, arterial hypertension, etc. PMID:19267666

  16. Box graphs and resolutions I

    NASA Astrophysics Data System (ADS)

    Braun, Andreas P.; Schäfer-Nameki, Sakura

    2016-04-01

    Box graphs succinctly and comprehensively characterize singular fibers of elliptic fibrations in codimension two and three, as well as flop transitions connecting these, in terms of representation theoretic data. We develop a framework that provides a systematic map between a box graph and a crepant algebraic resolution of the singular elliptic fibration, thus allowing an explicit construction of the fibers from a singular Weierstrass or Tate model. The key tool is what we call a fiber face diagram, which shows the relevant information of a (partial) toric triangulation and allows the inclusion of more general algebraic blowups. We shown that each such diagram defines a sequence of weighted algebraic blowups, thus providing a realization of the fiber defined by the box graph in terms of an explicit resolution. We show this correspondence explicitly for the case of SU (5) by providing a map between box graphs and fiber faces, and thereby a sequence of algebraic resolutions of the Tate model, which realizes each of the box graphs.

  17. Dead or alive: Deoxyribonuclease I sensitive bacteria and implications for the sinus microbiome

    PubMed Central

    Willis, Amanda L.; Calton, Joshua B.; Carr, Tara F.; Chiu, Alexander G.

    2016-01-01

    Background: Recently, there has been tremendous interest in the sinus microbiome and how it relates to disease. However, a lack of a standardized sample collection and DNA extraction methods makes comparison of results across studies nearly impossible. Furthermore, current techniques fail to identify which components of the microbiome are actually alive within the host at the time of sampling. Objective: To develop and optimize a method to differentiate which bacterial species in the human sinus microbiome are live versus dead. Methods: Duplicate samples from the middle meatus of patients with healthy sinus tissue and those patients with chronic rhinosinusitis were collected by using brushes (n = 12), swabs (n = 27), and tissue biopsy (n = 8) methods. One sample from each pair was either deoxyribonuclease I- or control-treated before DNA extraction. The relative bacterial versus human composition of each sample was determined. A 16S ribosomal RNA gene analysis was performed on a six-paired sample from patients with healthy sinus tissue. Results: We found that swabs and brushes collected a higher percentage of bacterial DNA than did tissue biopsy. We also determined that as much as 50% of the bacteria collected in these samples was already dead at the time of collection. The 16S ribosomal RNA gene analysis found significant changes in the relative abundance of taxa identified in the live versus dead bacterial communities of healthy human sinuses. Conclusions: Our findings indicated that swabs provided the best quality microbiome samples and that a large portion of the bacteria identified in the sinus were deoxyribonuclease I sensitive. These results highlighted the need for improved techniques such as those presented here, which can differentiate between living and dead bacteria in a sample, a potentially critical distinction when examining changes in sinus innate immune function because both components play important, but distinct, functions. Further studies will

  18. Asymptotic symmetries from finite boxes

    NASA Astrophysics Data System (ADS)

    Andrade, Tomás; Marolf, Donald

    2016-01-01

    It is natural to regulate an infinite-sized system by imposing a boundary condition at finite distance, placing the system in a 'box.' This breaks symmetries, though the breaking is small when the box is large. One should thus be able to obtain the asymptotic symmetries of the infinite system by studying regulated systems. We provide concrete examples in the context of Einstein-Hilbert gravity (with negative or zero cosmological constant) by showing in 4 or more dimensions how the anti-de Sitter and Poincaré asymptotic symmetries can be extracted from gravity in a spherical box with Dirichlet boundary conditions. In 2 + 1 dimensions we obtain the full double-Virasoro algebra of asymptotic symmetries for AdS3 and, correspondingly, the full Bondi-Metzner-Sachs (BMS) algebra for asymptotically flat space. In higher dimensions, a related approach may continue to be useful for constructing a good asymptotically flat phase space with BMS asymptotic symmetries.

  19. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  20. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  1. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  2. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  3. 9 CFR 314.8 - Dead animal carcasses.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Dead animal carcasses. 314.8 Section 314.8 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... Dead animal carcasses. (a) With the exception of dead livestock which have died en route and...

  4. 14 CFR 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Use of deadly force. 1203b.106 Section 1203b... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. Deadly force shall be used only in those circumstances where the security force officer reasonably believes...

  5. 14 CFR 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Use of deadly force. 1203b.106 Section... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. Deadly force shall be used only in those circumstances where the security force officer reasonably believes...

  6. Preventing Deadly Conflict: Toward a World without War.

    ERIC Educational Resources Information Center

    Francis, Greg

    Although some people believed that the end of the Cold War would herald a new age of peace, the 1990s have seen more than five million people die in over 35 deadly conflicts. New technologies have made warfare ever more deadly. There is, however, a breadth of options available to prevent or control deadly conflict in the world. This curriculum…

  7. 14 CFR 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 5 2012-01-01 2012-01-01 false Use of deadly force. 1203b.106 Section... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. Deadly force shall be used only in those circumstances where the security force officer reasonably believes...

  8. 14 CFR § 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 5 2014-01-01 2014-01-01 false Use of deadly force. § 1203b.106 Section Â... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. NASA security force personnel may use deadly force only when necessary, that is, when the officer has a...

  9. 14 CFR 1203b.106 - Use of deadly force.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 5 2013-01-01 2013-01-01 false Use of deadly force. 1203b.106 Section... AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.106 Use of deadly force. Deadly force shall be used only in those circumstances where the security force officer reasonably believes...

  10. Dead Sea Minerals loaded polymeric nanoparticles.

    PubMed

    Dessy, Alberto; Kubowicz, Stephan; Alderighi, Michele; Bartoli, Cristina; Piras, Anna Maria; Schmid, Ruth; Chiellini, Federica

    2011-10-15

    Therapeutic properties of Dead Sea Water (DSW) in the treatment of skin diseases such as atopic dermatitis, psoriasis and photo aging UV damaged skin have been well established. DSW is in fact rich in minerals such as calcium, magnesium, sodium, potassium, zinc and strontium which are known to exploit anti-inflammatory effects and to promote skin barrier recovery. In order to develop a Dead Sea Minerals (DSM) based drug delivery system for topical therapy of skin diseases, polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with monomethoxy poly(ethyleneglycol) 2000 MW (PEG) and 95% grafted with 2-methoxyethanol (VAM41-PEG) loaded with DSM were prepared by means of a combined miniemulsion/solvent evaporation process. The resulting nanoparticles were characterized in terms of dimension, morphology, biocompatibility, salt content and release. Cytocompatible spherical nanoparticles possessing an average diameter of about 300 nm, a time controlled drug release profile and a high formulation yield were obtained.

  11. Potential Evaporite Biomarkers from the Dead Sea

    NASA Technical Reports Server (NTRS)

    Morris, Penny A.; Wentworth, Susan J.; Thomas-Keprta, Kathie; Allen, Carlton C.; McKay, David S.

    2001-01-01

    The Dead Sea is located on the northern branch of the African-Levant Rift systems. The rift system, according to one model, was formed by a series of strike slip faults, initially forming approximately two million years ago. The Dead Sea is an evaporite basin that receives freshwater from springs and from the Jordan River. The Dead Sea is different from other evaporite basins, such as the Great Salt Lake, in that it possesses high concentrations of magnesium and has an average pH of 6.1. The dominant cation in the Great Salt Lake is sodium, and the pH is 7.7. Calcium concentrations are also higher in the Dead Sea than in the Great Salt Lake. Both basins are similar in that the dominant anion is chlorine and the salinity levels are approximately 20 %. Other common cations that have been identified from the waters of the Dead Sea and the Great Salt Lake include sodium and potassium. A variety of Archea, Bacteria, and a single genus of a green algal, Dunaliella, has been described from the Dead Sea. Earlier studies concentrated on microbial identification and analysis of their unique physiology that allows them to survive in this type of extreme environment. Potential microbial fossilization processes, microbial fossils, and the metallic ions associated with fossilization have not been studied thoroughly. The present study is restricted to identifying probable microbial morphologies and associated metallic ions. XRD (X Ray Diffraction) analysis indicates the presence of halite, quartz, and orthoclase feldspar. In addition to these minerals, other workers have reported potassium chloride, magnesium bromide, magnesium chloride, calcium chloride, and calcium sulfate. Halite, calcium sulfate, and orthoclase were examined in this report for the presence of microbes, microbially induced deposits or microbial alteration. Neither the gypsum nor the orthoclase surfaces possesses any obvious indications of microbial life or fossilization. The sand-sized orthoclase particles are

  12. Perturbative search for dead-end CFTs

    NASA Astrophysics Data System (ADS)

    Nakayama, Yu

    2015-05-01

    To explore the possibility of self-organized criticality, we look for CFTs without any relevant scalar deformations (a.k.a. dead-end CFTs) within power-counting renormalizable quantum field theories with a weakly coupled Lagrangian description. In three dimensions, the only candidates are pure (Abelian) gauge theories, which may be further deformed by Chern-Simons terms. In four dimensions, we show that there are infinitely many non-trivial candidates based on chiral gauge theories. Using the three-loop beta functions, we compute the gap of scaling dimensions above the marginal value, and it can be as small as and robust against the perturbative corrections. These classes of candidates are very weakly coupled and our perturbative conclusion seems difficult to refute. Thus, the hypothesis that non-trivial dead-end CFTs do not exist is likely to be false in four dimensions.

  13. Zero dead volume tube to surface seal

    DOEpatents

    Benett, William J.; Folta, James A.

    2000-01-01

    A method and apparatus for connecting a tube to a surface that creates a dead volume seal. The apparatus is composed of three components, a body, a ferrule, and a threaded fitting. The ferrule is compressed onto a tube and a seal is formed between the tube and a device retained in the body by threading the fitting into the body which provides pressure that seals the face of the ferrule to a mating surface on the device. This seal can be used at elevated temperatures depending on the materials used. While the invention has been developed for use with micro-machined silicon wafers used in Capillary Gas Chromatograph (GC), it can be utilized anywhere for making a gas or fluid face seal to the surface of a device that has near zero dead volume.

  14. Improving measurement of Chesapeake Bay's dead zone

    NASA Astrophysics Data System (ADS)

    Schultz, Colin

    2013-09-01

    In the 1930s, researchers first noticed that the Chesapeake Bay had a dead zone, an expanse of water with drastically reduced concentrations of oxygen. In the 1980s, hypoxia—low-oxygen conditions—gave way in some places to anoxia—a near-total depletion of dissolved oxygen. A lack of oxygen makes the water inhospitable for many marine organisms, and the Chesapeake Bay is the focus of major ecosystem rehabilitation efforts.

  15. Surviving deadness in the analytic experience.

    PubMed

    Koritar, Endre

    2014-12-01

    The transference/countertransference (third space) analysis is considered to be central in the therapeutic effectiveness of the analytic process. Less emphasis has been placed on the actual experiences of analyst and analysand in the conflictual reenactment of third space experience and its resolution. This paper recounts the shared experience of a patient who was silent throughout most of the analysis, and my reaction, in fantasy and enactment, to this disturbing experience-both for him and for myself. I argue that it is the affective re-experiencing of past repressed trauma in the analytic space that has a therapeutic impact, leading to growth in the patient and also the therapist. I contrast Freud's emphasis on insight, making the unconscious conscious, with Ferenczi's suggestion that the therapeutic impact lies in the repetition of past traumatic experience in the analysis but with the possibility of a different outcome with a more benign object, leading to symbolic representation of repressed trauma. Re-experiencing and symbolization, in the third space, of past traumatic experience can be an exit point from the endless repetition of trauma in internal and external object relations, leading to a new beginning in the patient's life. Immersed in the experience of deadness in the analysis, which had become a dead womb, the struggle to remain alive and thinking led to a rupture out of the dead womb, like the Caesura of birth, into aliveness and the ability to mentalize what had previously remained unmentalized. PMID:25434889

  16. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

    PubMed Central

    Zampedri, Cecilia; Tinoco-Cuellar, Maryana; Carrillo-Rosas, Samantha; Diaz-Tellez, Abigail; Ramos-Balderas, Jose Luis; Pelegri, Francisco

    2016-01-01

    ABSTRACT Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b) during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment. PMID:27489304

  17. Affinity Capture and Identification of Host Cell Factors Associated with Hepatitis C Virus (+) Strand Subgenomic RNA*

    PubMed Central

    Upadhyay, Alok; Dixit, Updesh; Manvar, Dinesh; Chaturvedi, Nootan; Pandey, Virendra N.

    2013-01-01

    Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3–4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication. PMID:23429521

  18. Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora

    PubMed Central

    Bollmann, Stephanie R.; Fang, Yufeng; Press, Caroline M.; Tyler, Brett M.; Grünwald, Niklaus J.

    2016-01-01

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed. PMID:27014308

  19. Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora.

    PubMed

    Bollmann, Stephanie R; Fang, Yufeng; Press, Caroline M; Tyler, Brett M; Grünwald, Niklaus J

    2016-01-01

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed. PMID:27014308

  20. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DIS- EASE (END) AND CHLAMYDIOSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  1. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DIS- EASE (END) AND CHLAMYDIOSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  2. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DISEASE (END) AND CHLAMYDI-OSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  3. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS NEWCASTLE DISEASE AND CHLAMYDIOSIS Newcastle Disease § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except as provided in paragraph (b)...

  4. 9 CFR 82.6 - Interstate movement of dead birds and dead poultry from a quarantined area.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... dead poultry from a quarantined area. 82.6 Section 82.6 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS EXOTIC NEWCASTLE DISEASE (END) AND CHLAMYDI-OSIS Exotic Newcastle Disease (END) § 82.6 Interstate movement of dead birds and dead poultry from a quarantined area. (a) Except...

  5. Vertical Mixing in the Dead Sea

    NASA Astrophysics Data System (ADS)

    Gertman, Isaac; Ozer, Tal; Katsenelson, Boris; Lensky, Nadav

    2015-04-01

    For hundreds of years, the Dead Sea was characterized by a stable haline stratification, supported by runoff. The penetration of the winter convection was limited to an upper mixed layer (UML) of about 30-50 m. Below the UML, a stable halocline prevented the mixing. As a result of the runoff reduction, the UML salinity increased and the gravitational stability diminished. During the winter of 1978-1979, the sea water overturned, ending the long-term stable hydrological regime. Since 1979, the haline stratification structure reoccurred twice after extremely rainy winters, in 1980-82 and 1992-1995. In other years, the sea was entirely mixed by winter thermal convection ( which occurs from November to March ) and had a seasonal pycnocline beneath the UML during summer. Profiles of temperature and quasi-salinity (density anomaly from 1000 kg/m3 for the chosen reference temperature of 32° C) during the last 19 years, show the formation of summer ``overturning halocline'' beneath the UML, and the thermocline that supports the stable stratification. Another warm and saline layer is formed also during the summer period near the bottom. This layer spreads from the southern part of the sea, where end-brine is discharged to the sea from the Israeli and Jordanian salt plants' evaporation ponds. The end-brine has extremely high salinity (˜ 350 g/kg) and, in spite of the high temperatures ( ˜ 45° C), high density (1350 kg/m^3), it therefore spreads as a gravitational current in the Dead Sea deep basin. Estimation of the density ratio (Rρ) for the Dead Sea water (where measurements of water salinity is quite difficult) was done using quasi-salinity (σ32) and potential temperature (θ): Rρ= [α(partialθ/partial z)]/[β(partial σ32/partial z)], where α and β are temperature expansion and quasi-salinity contraction coefficients respectively. The values of α and β for the Dead Sea water were defined from water samples collected during 2008. The Rρ values confirm that

  6. Nucleolar localization elements in U8 snoRNA differ from sequences required for rRNA processing.

    PubMed Central

    Lange, T S; Borovjagin, A V; Gerbi, S A

    1998-01-01

    U8 small nucleolar RNA (snoRNA) is essential for metazoan ribosomal RNA (rRNA) processing in nucleoli. The sequences and structural features in Xenopus U8 snoRNA that are required for its nucleolar localization were analyzed. Fluorescein-labeled U8 snoRNA was injected into Xenopus oocyte nuclei, and fluorescence microscopy of nucleolar preparations revealed that wild-type Xenopus U8 snoRNA localized to nucleoli, regardless of the presence or nature of the 5' cap on the injected U8 snoRNA. Nucleolar localization was observed when loops or stems in the 5' portion of U8 that are critical for U8 snoRNA function in rRNA processing were mutated. Therefore, sites of interaction in U8 snoRNA that potentially tether it to pre-rRNA are not essential for nucleolar localization of U8. Boxes C and D are known to be nucleolar localization elements (NoLEs) for U8 snoRNA and other snoRNAs of the Box C/D family. However, the spatial relationship of Box C to Box D was not crucial for U8 nucleolar localization, as demonstrated here by deletion of sequences in the two stems that separate them. These U8 mutants can localize to nucleoli and function in rRNA processing as well. The single-stranded Cup region in U8, adjacent to evolutionarily conserved Box C, functions as a NoLE in addition to Boxes C and D. Cup is unique to U8 snoRNA and may help bind putative protein(s) needed for nucleolar localization. Alternatively, Cup may help to retain U8 snoRNA within the nucleolus. PMID:9671052

  7. Trapping solids at the inner edge of the dead zone: 3-D global MHD simulations

    NASA Astrophysics Data System (ADS)

    Dzyurkevich, N.; Flock, M.; Turner, N. J.; Klahr, H.; Henning, Th.

    2010-06-01

    Context. The poorly-ionized interior of the protoplanetary disk or “dead zone” is the location where dust coagulation processes may be most efficient. However even here, planetesimal formation may be limited by the loss of solid material through radial drift, and by collisional fragmentation of the particles. Both depend on the turbulent properties of the gas. Aims: Our aim here is to investigate the possibility that solid particles are trapped at local pressure maxima in the dynamically evolving disk. We perform the first 3-D global non-ideal magnetohydrodynamical (MHD) calculations of a section of the disk treating the turbulence driven by the magneto-rotational instability (MRI). Methods: We use the ZeusMP code with a fixed Ohmic resistivity distribution. The domain contains an inner MRI-active region near the young star and an outer midplane dead zone, with the transition between the two modeled by a sharp increase in the magnetic diffusivity. Results: The azimuthal magnetic fields generated in the active zone oscillate over time, changing sign about every 150 years. We thus observe the radial structure of the “butterfly pattern” seen previously in local shearing-box simulations. The mean magnetic field diffuses from the active zone into the dead zone, where the Reynolds stress nevertheless dominates, giving a residual α between 10-4 and 10-3. The greater total accretion stress in the active zone leads to a net reduction in the surface density, so that after 800 years an approximate steady state is reached in which a local radial maximum in the midplane pressure lies near the transition radius. We also observe the formation of density ridges within the active zone. Conclusions: The dead zone in our models possesses a mean magnetic field, significant Reynolds stresses and a steady local pressure maximum at the inner edge, where the outward migration of planetary embryos and the efficient trapping of solid material are possible.

  8. The loop structure and the RNA helicase p72/DDX17 influence the processing efficiency of the mice miR-132.

    PubMed

    Remenyi, Judit; Bajan, Sarah; Fuller-Pace, Frances V; Arthur, J Simon C; Hutvagner, Gyorgy

    2016-01-01

    miRNAs are small RNAs that are key regulators of gene expression in eukaryotic organisms. The processing of miRNAs is regulated by structural characteristics of the RNA and is also tightly controlled by auxiliary protein factors. Among them, RNA binding proteins play crucial roles to facilitate or inhibit miRNA maturation and can be controlled in a cell, tissue and species-specific manners or in response to environmental stimuli. In this study we dissect the molecular mechanism that promotes the overexpression of miR-132 in mice over its related, co-transcribed and co-regulated miRNA, miR-212. We have shown that the loop structure of miR-132 is a key determinant for its efficient processing in cells. We have also identified a range of RNA binding proteins that recognize the loop of miR-132 and influence both miR-132 and miR-212 processing. The DEAD box helicase p72/DDX17 was identified as a factor that facilitates the specific processing of miR-132. PMID:26947125

  9. NETL's JIC in a box

    ScienceCinema

    David Anna

    2016-07-12

    The National Energy Technology Laboratory developed the idea of a portable joint information center AKA JIC in-a-box. This video discribes some of the equipment in the portable JIC as well as some of the methodology that NETL developed as a result of this portable JIC concept.

  10. The Cereal Box Problem Revisited.

    ERIC Educational Resources Information Center

    Wilkins, Jesse L. M.

    1999-01-01

    Investigates the cereal box problem using both an experimental and theoretical framework, and Monte Carlo methods. Using empirical data, students can discover patterns and relationships that help them understand the origin of the theoretical solution to the problem. Contains 17 references. (Author/ASK)

  11. NETL's JIC in a box

    SciTech Connect

    David Anna

    2009-06-03

    The National Energy Technology Laboratory developed the idea of a portable joint information center AKA JIC in-a-box. This video discribes some of the equipment in the portable JIC as well as some of the methodology that NETL developed as a result of this portable JIC concept.

  12. Black Boxes in Workplace Mathematics

    ERIC Educational Resources Information Center

    Williams, Julian; Wake, Geoff

    2007-01-01

    We ground Cultural-Historical Activity Theory (CHAT) in studies of workplace practices from a mathematical point of view. We draw on multiple case study visits by college students and teacher-researchers to workplaces. By asking questions that "open boxes", we "outsiders and boundary-crossers" sought to expose contradictions between College and…

  13. On the Dirichlet's Box Principle

    ERIC Educational Resources Information Center

    Poon, Kin-Keung; Shiu, Wai-Chee

    2008-01-01

    In this note, we will focus on several applications on the Dirichlet's box principle in Discrete Mathematics lesson and number theory lesson. In addition, the main result is an innovative game on a triangular board developed by the authors. The game has been used in teaching and learning mathematics in Discrete Mathematics and some high schools in…

  14. The Bird Box Survey Project

    ERIC Educational Resources Information Center

    Willis, Patrick

    2014-01-01

    When high school students are asked what's the best part of science class, many will say it's the field trips. Students enjoy engaging in authentic, community-based science outside the classroom. To capitalize on this, Patrick Willis created the Bird Box Survey Project for his introductory field biology class. The project takes students…

  15. Evaluation of bike boxes at signalized intersections.

    PubMed

    Dill, Jennifer; Monsere, Christopher M; McNeil, Nathan

    2012-01-01

    This paper presents a before-after study of bike boxes at 10 signalized intersections in Portland, Oregon. The bike boxes, also known as advanced stop lines or advanced stop boxes, were installed to increase visibility of cyclists and reduce conflicts between motor vehicles and cyclists, particularly in potential "right-hook" situations. Before and after video were analyzed for seven intersections with green bike boxes, three intersections with uncolored bike boxes, and two control intersections. User perceptions were measured through surveys of cyclists passing through five of the bike box intersections and of motorists working downtown, where the boxes were concentrated. Both the observations and survey of motorists found a high rate of compliance and understanding of the markings. Overall, 73% of the stopping motor vehicles did not encroach at all into the bike box. Both motor vehicle and bicycle encroachment in the pedestrian crosswalk fell significantly at the bike box locations compared to the control intersections. The bike boxes had mixed effects on the motorists' encroachment in the bicycle lane. The number of observed conflicts at the bike box locations decreased, while the total number of cyclists and motor vehicles turning right increased. Negative-binomial models based upon the data predict fewer conflicts with the boxes, particularly as right-turning motor vehicle volumes increase. Observations of yielding behavior at two bike box and one control intersection found an improvement in motorists yielding to cyclists at the bike box locations. Differences in the traffic volumes and location contexts make firm conclusions about the effects of green coloring of the boxes difficult. Higher shares of surveyed motorists felt that the bike boxes made driving safer rather than more dangerous, even when the sample was narrowed to respondents who were not also cyclists. Over three-quarters of the surveyed cyclists thought that the boxes made the intersection safer.

  16. Comet 'Bites the Dust' Around Dead Star

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Infrared Spectrometer Graph

    This artist's concept illustrates a comet being torn to shreds around a dead star, or white dwarf, called G29-38. NASA's Spitzer Space Telescope observed a cloud of dust around this white dwarf that may have been generated from this type of comet disruption. The findings suggest that a host of other comet survivors may still orbit in this long-dead solar system.

    The white dwarf G29-38 began life as a star that was about three times as massive as our sun. Its death involved the same steps that the sun will ultimately undergo billions of years from now. According to theory, the G29-38 star became brighter and brighter as it aged, until it bloated up into a dying star called a red giant. This red giant was large enough to engulf and evaporate any terrestrial planets like Earth that happened to be in its way. Later, the red giant shed its outer atmosphere, leaving behind a shrunken skeleton of star, called a white dwarf. If the star did host a planetary system, outer planets akin to Jupiter and Neptune and a remote ring of icy comets would remain.

    The Spitzer observations provide observational evidence for this orbiting outpost of comet survivors. Astronomers speculate that one such comet was knocked into the inner regions of G29-38, possibly by an outer planet. As the comet approached very close to the white dwarf, it may have been torn apart by the star's tidal forces. Eventually, all that would be left of the comet is a disk of dust.

    This illustration shows a comet in the process of being pulverized: part of it still exists as a chain of small clumps, while the rest has already spread out into a dusty disk. Comet Shoemaker-Levy 9 broke apart in a similar fashion when it plunged into Jupiter in 1994. Evidence for Comets Found in Dead Star's Dust The graph of data, or spectrum, from NASA's Spitzer Space Telescope indicates that a dead star, or white dwarf, called G29

  17. The five deadly sins of science publishing

    PubMed Central

    Tracz, Vitek

    2015-01-01

    Science cannot progress without scientists reporting their findings. And yet researchers have given control of this central pillar of the scientific process to science publishers, who are in the business of serving the interests of their journals; these are not always the same as the interests of science. This editorial describes the problems with the process of preparing and publishing research findings, and with judging their veracity and significance, and then explains how we at Faculty of 1000 are starting to tackle the ‘deadly sins’ of science publishing. PMID:26097694

  18. [A case of lycanthropy with deadly violence].

    PubMed

    Bénézech, M; De Witte, J; Etchepare, J J; Bourgeois, M

    1989-01-01

    After a short historical review of the contemporary medical literature, the authors analyze a new and original observation of lycanthropy. He is a 28 years old man, imprisoned for deadly violence, who has been showing, for many years, the belief of being transformed into a werewolf during depersonalization episodes when he presents a lycanthropic behaviour. Our observation is closer to hysteria and mythomania on an antisocial personality, although it seems difficult to place the mental pathology of this alcoholic recidivist delinquent into a nosographical frame.

  19. Glove box for water pit applications

    DOEpatents

    Mills, William C.; Rabe, Richard A.

    2005-01-18

    A glove box assembly that includes a glove box enclosure attached to a longitudinally extending hollow tube having an entranceway, wherein the portion of the tube is in a liquid environment. An elevator member is provided for raising an object that is introduced into the hollow tube from the liquid environment to a gas environment inside the glove box enclosure while maintaining total containment.

  20. The Guide to the Ecology Box.

    ERIC Educational Resources Information Center

    Ontario Inst. for Studies in Education, Toronto.

    Cooperating with the Canadian Commission for UNESCO, the Ontario Institute for Studies in Education has prepared boxes of experimental curriculum materials on the subject of ecology. This guide summarizes the design and contents of the boxes and provides instructions for those using the boxes--principals, teachers, parents, librarians, and…

  1. The Heuristic Interpretation of Box Plots

    ERIC Educational Resources Information Center

    Lem, Stephanie; Onghena, Patrick; Verschaffel, Lieven; Van Dooren, Wim

    2013-01-01

    Box plots are frequently used, but are often misinterpreted by students. Especially the area of the box in box plots is often misinterpreted as representing number or proportion of observations, while it actually represents their density. In a first study, reaction time evidence was used to test whether heuristic reasoning underlies this…

  2. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  3. Illumination box and camera system

    DOEpatents

    Haas, Jeffrey S.; Kelly, Fredrick R.; Bushman, John F.; Wiefel, Michael H.; Jensen, Wayne A.; Klunder, Gregory L.

    2002-01-01

    A hand portable, field-deployable thin-layer chromatography (TLC) unit and a hand portable, battery-operated unit for development, illumination, and data acquisition of the TLC plates contain many miniaturized features that permit a large number of samples to be processed efficiently. The TLC unit includes a solvent tank, a holder for TLC plates, and a variety of tool chambers for storing TLC plates, solvent, and pipettes. After processing in the TLC unit, a TLC plate is positioned in a collapsible illumination box, where the box and a CCD camera are optically aligned for optimal pixel resolution of the CCD images of the TLC plate. The TLC system includes an improved development chamber for chemical development of TLC plates that prevents solvent overflow.

  4. Ethics of Practicing Medical Procedures on Newly Dead and Nearly Dead Patients

    PubMed Central

    Berger, Jeffrey T; Rosner, Fred; Cassell, Eric J

    2002-01-01

    OBJECTIVE To examine the ethical issues raised by physicians performing, for skill development, medically nonindicated invasive medical procedures on newly dead and dying patients. DESIGN Literature review; issue analysis employing current normative ethical obligations, and evaluation against moral rules and utilitarian assessments manifest in other common perimortem practices. RESULTS Practicing medical procedures for training purposes is not uncommon among physicians in training. However, empiric information is limited or absent evaluating the effects of this practice on physician competence and ethics, assessing public attitudes toward practicing medical procedures and requirements for consent, and discerning the effects of a consent requirement on physicians' clinical competence. Despite these informational gaps, there is an obligation to secure consent for training activities on newly and nearly dead patients based on contemporary norms for informed consent and family respect. Paradigms of consent-dependent societal benefits elsewhere in health care support our determination that the benefits from physicians practicing procedures does not justify setting aside the informed consent requirement. CONCLUSION Current ethical norms do not support the practice of using newly and nearly dead patients for training in invasive medical procedures absent prior consent by the patient or contemporaneous surrogate consent. Performing an appropriately consented training procedure is ethically acceptable when done under competent supervision and with appropriate professional decorum. The ethics of training on the newly and nearly dead remains an insufficiently examined area of medical training. PMID:12390553

  5. Asteroid 'Bites the Dust' Around Dead Star

    NASA Technical Reports Server (NTRS)

    2009-01-01

    NASA's Spitzer Space Telescope set its infrared eyes upon the dusty remains of shredded asteroids around several dead stars. This artist's concept illustrates one such dead star, or 'white dwarf,' surrounded by the bits and pieces of a disintegrating asteroid. These observations help astronomers better understand what rocky planets are made of around other stars.

    Asteroids are leftover scraps of planetary material. They form early on in a star's history when planets are forming out of collisions between rocky bodies. When a star like our sun dies, shrinking down to a skeleton of its former self called a white dwarf, its asteroids get jostled about. If one of these asteroids gets too close to the white dwarf, the white dwarf's gravity will chew the asteroid up, leaving a cloud of dust.

    Spitzer's infrared detectors can see these dusty clouds and their various constituents. So far, the telescope has identified silicate minerals in the clouds polluting eight white dwarfs. Because silicates are common in our Earth's crust, the results suggest that planets similar to ours might be common around other stars.

  6. Leishmania infantum LeIF protein is an ATP-dependent RNA helicase and an eIF4A-like factor that inhibits translation in yeast.

    PubMed

    Barhoumi, Mourad; Tanner, N K; Banroques, Josette; Linder, Patrick; Guizani, Ikram

    2006-11-01

    LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast. PMID:17087726

  7. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  8. Naf1 p is a box H/ACA snoRNP assembly factor.

    PubMed Central

    Fatica, Alessandro; Dlakić, Mensur; Tollervey, David

    2002-01-01

    Box H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) contain four essential proteins, Cbf5p, Gar1p, Nhp2p, and Nop10p, each of which, with the exception of Gar1p, is required for box H/ACA snoRNA accumulation. Database searches identified a novel essential protein, which we termed Naf1p, with a region of homology to the RNA-binding domain of Gar1p and other features in common with hnRNP-like proteins. Naf1p is localized to the nucleus and is not a stable component of the H/ACA snoRNPs, but it is required for the accumulation of all box H/ACA snoRNAs. This requirement is not at the level of snoRNA transcription initiation or termination. Naf1 p shows in vitro RNA-binding activity and also binds directly to Cbf5p and Nhp2p. Naf1p was shown to bind to the CTD in vivo in a two-hybrid assay, and the phosphorylated CTD, but not the nonphosphorylated CTD, was shown to precipitate tagged Naf1p from a cell lysate. We propose that Naf1 p is recruited to the CTD of RNA polymerase II and binds to nascent box H/ACA snoRNAs promoting snoRNP assembly. PMID:12515383

  9. Photographic Study Of A Dead-Pressed Explosive

    NASA Astrophysics Data System (ADS)

    Swallowe, G. M.; Field, J. E.

    1983-03-01

    High speed photography in conjunction with electron microscopy and a pressure measuring technique have been used to investigate the differences between dead-pressed and non-dead-pressed samples of the primary explosive Mercury Fulminate (Hg Ful). Photographs of reaction propagation were taken in transmitted light using a specially adapted drop-weight machine with transparent anvils. The results of these experiments suggested a mechanism for dead-pressing in Hg Ful based on the microscopic internal structure of the compacted explosive.

  10. Turbulence, Transport, and Waves in Ohmic Dead Zones

    NASA Astrophysics Data System (ADS)

    Gole, Daniel; Simon, Jacob B.; Lubow, Stephen H.; Armitage, Philip J.

    2016-07-01

    We use local numerical simulations to study a vertically stratified accretion disk with a resistive mid-plane that damps magnetohydrodynamic (MHD) turbulence. This is an idealized model for the dead zones that may be present at some radii in protoplanetary and dwarf novae disks. We vary the relative thickness of the dead and active zones to quantify how forced fluid motions in the dead zone change. We find that the residual Reynolds stress near the mid-plane decreases with increasing dead zone thickness, becoming negligible in cases where the active to dead mass ratio is less than a few percent. This implies that purely Ohmic dead zones would be vulnerable to episodic accretion outbursts via the mechanism of Martin & Lubow. We show that even thick dead zones support a large amount of kinetic energy, but this energy is largely in fluid motions that are inefficient at angular momentum transport. Confirming results from Oishi & Mac Low, the perturbed velocity field in the dead zone is dominated by an oscillatory, vertically extended circulation pattern with a low frequency compared to the orbital frequency. This disturbance has the properties predicted for the lowest order r mode in a hydrodynamic disk. We suggest that in a global disk similar excitations would lead to propagating waves, whose properties would vary with the thickness of the dead zone and the nature of the perturbations (isothermal or adiabatic). Flows with similar amplitudes would buckle settled particle layers and could reduce the efficiency of pebble accretion.

  11. The ecosystem service value of living versus dead biogenic reef

    NASA Astrophysics Data System (ADS)

    Sheehan, E. V.; Bridger, D.; Attrill, M. J.

    2015-03-01

    Mixed maerl beds (corralline red algae) comprise dead thalli with varying amounts of live maerl fragments, but previously it was not known whether the presence of the live maerl increases the ecosystem service 'habitat provision' of the dead maerl for the associated epibenthos. A 'flying array' towed sled with high definition video was used to film transects of the epibenthos in dead maerl and mixed maerl beds in two locations to the north and south of the English Channel (Falmouth and Jersey). Mixed maerl beds supported greater number of taxa and abundance than dead beds in Falmouth, while in Jersey, mixed and dead beds supported similar number of taxa and dead beds had a greater abundance of epifauna. Scallops tended to be more abundant on mixed beds than dead beds. Tube worms were more abundant on mixed beds in Falmouth and dead beds in Jersey. An increasing percentage occurrence of live maerl thalli correlated with increasing number of taxa in Falmouth but not Jersey. It was concluded that while live thalli can increase the functional role of dead maerl beds for the epibenthos, this is dependent on location and response variable. As a result of this work, maerl habitat in SE Jersey has been protected from towed demersal fishing gear.

  12. Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

    SciTech Connect

    Nithianantham, Stanley; Shilton, Brian H

    2011-09-28

    The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motif V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.

  13. Analysis of the Isolated SecA DEAD Motor Suggests a Mechanism for Chemical-Mechanical Coupling

    SciTech Connect

    Nithianantham, Stanley; Shilton, Brian H

    2010-09-20

    The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motif V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.

  14. HIV and Tuberculosis: a Deadly Human Syndemic

    PubMed Central

    Kwan, Candice K.; Ernst, Joel D.

    2011-01-01

    Summary: A syndemic is defined as the convergence of two or more diseases that act synergistically to magnify the burden of disease. The intersection and syndemic interaction between the human immunodeficiency virus (HIV) and tuberculosis (TB) epidemics have had deadly consequences around the world. Without adequate control of the TB-HIV syndemic, the long-term TB elimination target set for 2050 will not be reached. There is an urgent need for additional resources and novel approaches for the diagnosis, treatment, and prevention of both HIV and TB. Moreover, multidisciplinary approaches that consider HIV and TB together, rather than as separate problems and diseases, will be necessary to prevent further worsening of the HIV-TB syndemic. This review examines current knowledge of the state and impact of the HIV-TB syndemic and reviews the epidemiological, clinical, cellular, and molecular interactions between HIV and TB. PMID:21482729

  15. Lighting up a Dead Star's Layers

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This image from NASA's Spitzer Space Telescope shows the scattered remains of an exploded star named Cassiopeia A. Spitzer's infrared detectors 'picked' through these remains and found that much of the star's original layering had been preserved.

    In this false-color image, the faint, blue glow surrounding the dead star is material that was energized by a shock wave, called the forward shock, which was created when the star blew up. The forward shock is now located at the outer edge of the blue glow. Stars are also seen in blue. Green, yellow and red primarily represent material that was ejected in the explosion and heated by a slower shock wave, called the reverse shock wave.

    The picture was taken by Spitzer's infrared array camera and is a composite of 3.6-micron light (blue); 4.5-micron light (green); and 8.0-micron light (red).

  16. New hot box solar cooker

    SciTech Connect

    Wang Xiping; Hou Shuqin; Sha Yongling; Liu Zude

    1992-12-31

    At present, over 100,000 solar cookers are in service in China. Most of these are concentrating cookers, making use of reflectors to concentrate sunlight at the cooking area. These cookers offer higher efficiency, more power and shorter cooking times. Since 1975 the authors have researched solar energy applications and, specifically, solar cookers. The major work has been the development of design calculations, selection of structure and materials, and performance testing. This paper describes the testing of several collection surface structures and box structures.

  17. Voronoi Diagrams Without Bounding Boxes

    NASA Astrophysics Data System (ADS)

    Sang, E. T. K.

    2015-10-01

    We present a technique for presenting geographic data in Voronoi diagrams without having to specify a bounding box. The method restricts Voronoi cells to points within a user-defined distance of the data points. The mathematical foundation of the approach is presented as well. The cell clipping method is particularly useful for presenting geographic data that is spread in an irregular way over a map, as for example the Dutch dialect data displayed in Figure 2. The automatic generation of reasonable cell boundaries also makes redundant a frequently used solution to this problem that requires data owners to specify region boundaries, as in Goebl (2010) and Nerbonne et al (2011).

  18. Speaking to the dead: images of the dead in contemporary art.

    PubMed

    O'Neill, Mary

    2011-05-01

    In this article I explore works by three artists in which we can see images that relate to bereavement. In the work of the first two, Araya Rasdjarmrearnsook and Andres Serrano, we can see photographic images (still and moving) of human corpses, which have been criticized as morbid and unhealthy. However I argue that it is not in fact images of death or the dead that are problematic but those images which present or evoke evidence of the emotions associated with death, and create a situation where we imagine the circumstances of our own deaths or the death of those we love. Images of the dead are acceptable as long as they do not cause pain to the living, as in a video game fantasy or a fiction, or are seen as other and distant. In the second group of works, by Gustgav Metzger, The Absent Dead: The Surrogate Body, the body is not present either because the death has taken place at a distance, either in time or geographically, or both, and a new site must be created. In this section, I discuss Metzger's auto-destructive art and argue that these works, through their ephemerality, embody a form of 'meaning making' and a possibility of the benefits of grief as described by Parkes.

  19. Zombie Vortices: The Dead Zones of Protoplanetary Disks are Not Dead

    NASA Astrophysics Data System (ADS)

    Jiang, Chung-Hsiang; Marcus, Philip; Pei, Suyang; Barranco, Joe; Hassanzadeh, Pedram; Lecoanet, Daniel

    2014-11-01

    Numerical simulations, using both the anelastic and fully compressible equations of motion, show that the ``dead zones'' of protoplanetary disks (PPDs) around forming stars are unstable and filled with vortex-dominated turbulence with Mach and Rossby numbers of order 0.2 - 0.3. The dead zones are regions in which the temperature is too cool for the gas to ionize and be destabilized by instabilities associated with the magnetic field. The ``dead zones'' were thought, by most authors, to be stable to all purely-hydrodynamic instabilities because the flow has an angular momentum that increases with increasing radius in a PPD and is therefore stable by Rayleigh's theorem. However, that theorem in not applicable to stratified flows, such as those in a PPD. We summarize our simulations with emphasis on the finite-amplitude trigger of the instability and show that when the trigger is Kolmogorov noise, the Mach number of the noise that is needed to create instability is proportional to Re - 1 / 2 , where Re is the Reynolds number of the initial noise.

  20. Simulation of Simple Controlled Processes with Dead-Time.

    ERIC Educational Resources Information Center

    Watson, Keith R.; And Others

    1985-01-01

    The determination of closed-loop response of processes containing dead-time is typically not covered in undergraduate process control, possibly because the solution by Laplace transforms requires the use of Pade approximation for dead-time, which makes the procedure lengthy and tedious. A computer-aided method is described which simplifies the…

  1. Zero-dead-time operation of interleaved atomic clocks.

    PubMed

    Biedermann, G W; Takase, K; Wu, X; Deslauriers, L; Roy, S; Kasevich, M A

    2013-10-25

    We demonstrate a zero-dead-time operation of atomic clocks. This clock reduces sensitivity to local oscillator noise, integrating as nearly 1/τ whereas a clock with dead time integrates as 1/τ(1/2) under identical conditions. We contend that a similar scheme may be applied to improve the stability of optical clocks.

  2. Recipe for Hypoxia: Playing the Dead Zone Game

    ERIC Educational Resources Information Center

    Kastler, Jessica A.

    2009-01-01

    Dead zones--areas experiencing low levels of dissolved oxygen--are growing in shallow ocean waters around the world. Research has shown that dead zones form as a result of a specific type of pollution, called nutrient enrichment or eutrophication, and are found in almost every coastal zone where humans have large populations. Concepts related to…

  3. Zero-dead-time operation of interleaved atomic clocks.

    PubMed

    Biedermann, G W; Takase, K; Wu, X; Deslauriers, L; Roy, S; Kasevich, M A

    2013-10-25

    We demonstrate a zero-dead-time operation of atomic clocks. This clock reduces sensitivity to local oscillator noise, integrating as nearly 1/τ whereas a clock with dead time integrates as 1/τ(1/2) under identical conditions. We contend that a similar scheme may be applied to improve the stability of optical clocks. PMID:24206471

  4. 37 CFR 1.42 - When the inventor is dead.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false When the inventor is dead. 1.42 Section 1.42 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... for A Patent § 1.42 When the inventor is dead. In case of the death of the inventor, the...

  5. 37 CFR 1.422 - When the inventor is dead.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false When the inventor is dead. 1.422 Section 1.422 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE... File An International Application § 1.422 When the inventor is dead. In case of the death of...

  6. Day of the Dead: A Mexican-American Celebration.

    ERIC Educational Resources Information Center

    Hoyt-Goldsmith, Diane

    This children's book describes how a Mexican-American family celebrates the traditional Mexican holiday, Day of the Dead (Dia de Muertos). The book centers on 10-year-old twins, Ximena and Azucena, who live in Sacramento, California, with their two brothers, older sister, and parents. The Day of the Dead takes place on the first and second day of…

  7. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  8. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  9. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  10. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  11. 10 CFR 1047.7 - Use of deadly force.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... explosives). (3) Nuclear weapons or nuclear explosive devices. When deadly force reasonably appears to be necessary to prevent the theft, sabotage, or unauthorized control of a nuclear weapon or nuclear explosive device. (4) Special nuclear material. When deadly force reasonably appears to be necessary to prevent...

  12. 37 CFR 1.422 - When the inventor is dead.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false When the inventor is dead. 1... File An International Application § 1.422 When the inventor is dead. In case of the death of the inventor, the legal representative (executor, administrator, etc.) of the deceased inventor may file...

  13. 37 CFR 1.42 - When the inventor is dead.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false When the inventor is dead. 1... for A Patent § 1.42 When the inventor is dead. In case of the death of the inventor, the legal representative (executor, administrator, etc.) of the deceased inventor may make the necessary oath...

  14. Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression.

    PubMed

    Snyder, Elizabeth; Soundararajan, Ramani; Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E

    2015-12-01

    The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2-/-;Ybx3-/- double mutants using a previously reported Ybx2-/- model and a newly generated global Ybx3-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.

  15. Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

    PubMed Central

    Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

    2015-01-01

    The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

  16. The Lithium Vapor Box Divertor

    NASA Astrophysics Data System (ADS)

    Goldston, Robert; Hakim, Ammar; Hammett, Gregory; Jaworski, Michael; Myers, Rachel; Schwartz, Jacob

    2015-11-01

    Projections of scrape-off layer width to a demonstration power plant suggest an immense parallel heat flux, of order 12 GW/m2, which will necessitate nearly fully detached operation. Building on earlier work by Nagayama et al. and by Ono et al., we propose to use a series of differentially pumped boxes filled with lithium vapor to isolate the buffering vapor from the main plasma chamber, allowing stable detachment. This powerful differential pumping is only available for condensable vapors, not conventional gases. We demonstrate the properties of such a system through conservation laws for vapor mass and enthalpy, and then include plasma entrainment and ultimately an estimate of radiated power. We find that full detachment should be achievable with little leakage of lithium to the main plasma chamber. We also present progress towards solving the Navier-Stokes equation numerically for the chain of vapor boxes, including self-consistent wall boundary conditions and fully-developed shocks, as well as concepts for an initial experimental demonstration-of-concept. This work supported by DOE Contract No. DE-AC02-09CH11466.

  17. Box graphs and singular fibers

    NASA Astrophysics Data System (ADS)

    Hayashi, Hirotaka; Lawrie, Craig; Morrison, David R.; Schafer-Nameki, Sakura

    2014-05-01

    We determine the higher codimension fibers of elliptically fibered Calabi-Yau fourfolds with section by studying the three-dimensional = 2 supersymmetric gauge theory with matter which describes the low energy effective theory of M-theory compactified on the associated Weierstrass model, a singular model of the fourfold. Each phase of the Coulomb branch of this theory corresponds to a particular resolution of the Weierstrass model, and we show that these have a concise description in terms of decorated box graphs based on the representation graph of the matter multiplets, or alternatively by a class of convex paths on said graph. Transitions between phases have a simple interpretation as "flopping" of the path, and in the geometry correspond to actual flop transitions. This description of the phases enables us to enumerate and determine the entire network between them, with various matter representations for all reductive Lie groups. Furthermore, we observe that each network of phases carries the structure of a (quasi-)minuscule representation of a specific Lie algebra. Interpreted from a geometric point of view, this analysis determines the generators of the cone of effective curves as well as the network of flop transitions between crepant resolutions of singular elliptic Calabi-Yau fourfolds. From the box graphs we determine all fiber types in codimensions two and three, and we find new, non-Kodaira, fiber types for E 6, E7 and E 8.

  18. Helicopter Gravity Survey in the Dead Sea Area

    NASA Astrophysics Data System (ADS)

    Götze, H.-J.; Meyer, Uwe; Choi, Sungchan

    2010-03-01

    Earthquakes have been reported in the Middle East since biblical times. Seismological studies demonstrate that many damaging earthquakes occurred along the Dead Sea Transform (DST), a long shear zone that extends from the Gulf of Aqaba/Eilat through the Dead Sea into the Sea of Galilee. To better understand the behavior and dynamics of the Dead Sea Transform, the Dead Sea Integrated Research (DESIRE) interdisciplinary and international project was initiated in 2006. The DST, which forms one of the world’s largest pull-apart basins (120 kilometers long, 20 kilometers wide, and about 10 kilometers deep), is a system of left-lateral strike-slip faults that accommodates about 105 kilometers of relative motion between the African and Arabian plates. To study the DST in places where it is easily accessible, the DESIRE project area focused on the Dead Sea basin (Figure 1).

  19. Dead-time compensation for a logarithmic display rate meter

    DOEpatents

    Larson, J.A.; Krueger, F.P.

    1987-10-05

    An improved circuit is provided for application to a radiation survey meter that uses a detector that is subject to dead time. The circuit compensates for dead time over a wide range of count rates by producing a dead-time pulse for each detected event, a live-time pulse that spans the interval between dead-time pulses, and circuits that average the value of these pulses over time. The logarithm of each of these values is obtained and the logarithms are subtracted to provide a signal that is proportional to a count rate that is corrected for the effects of dead time. The circuit produces a meter indication and is also capable of producing an audible indication of detected events. 5 figs.

  20. Dead-time compensation for a logarithmic display rate meter

    DOEpatents

    Larson, John A.; Krueger, Frederick P.

    1988-09-20

    An improved circuit is provided for application to a radiation survey meter that uses a detector that is subject to dead time. The circuit compensates for dead time over a wide range of count rates by producing a dead-time pulse for each detected event, a live-time pulse that spans the interval between dead-time pulses, and circuits that average the value of these pulses over time. The logarithm of each of these values is obtained and the logarithms are subtracted to provide a signal that is proportional to a count rate that is corrected for the effects of dead time. The circuit produces a meter indication and is also capable of producing an audible indication of detected events.

  1. Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes

    PubMed Central

    Ramesh, Arati; Wakeman, Catherine A.; Winkler, Wade C.

    2011-01-01

    The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses of other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base stacking, electrostatic, and hydrogen bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation-binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal-binding sites a manganese-chelated M-box RNA complex was resolved to 1.86 angstroms by X-ray crystallography. These data support the presence of at least 8 well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal binding cores within the M-box RNA that facilitate a network of long range interactions within the metal-bound, compacted conformation. PMID:21315082

  2. Insights into Metalloregulation by M-box Riboswitch RNAs via Structural Analysis of Manganese-Bound Complexes

    SciTech Connect

    Ramesh, Arati; Wakeman, Catherine A.; Winkler, Wade C.

    2011-12-09

    The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 {angstrom} by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.

  3. Interchangeable breech lock for glove boxes

    SciTech Connect

    Lemonds, David Preston

    2015-11-24

    A breech lock for a glove box is provided that may be used to transfer one or more items into the glove box. The breech lock can be interchangeably installed in place of a plug, glove, or other device in a port or opening of a glove box. Features are provided to aid the removal of items from the breech lock by a gloved operator. The breech lock can be reused or, if needed, can be replaced with a plug, glove, or other device at the port or opening of the glove box.

  4. Repackaging SRS Black Box TRU Waste

    SciTech Connect

    Swale, D. J.; Stone, K.A.; Milner, T. N.

    2006-01-09

    Historically, large items of TRU Waste, which were too large to be packaged in drums for disposal have been packaged in various sizes of custom made plywood boxes at the Savannah River Site (SRS), for many years. These boxes were subsequently packaged into large steel ''Black Boxes'' for storage at SRS, pending availability of Characterization and Certification capability, to facilitate disposal of larger items of TRU Waste. There are approximately 107 Black Boxes in inventory at SRS, each measuring some 18' x 12' x 7', and weighing up to 45,000 lbs. These Black Boxes have been stored since the early 1980s. The project to repackage this waste into Standard Large Boxes (SLBs), Standard Waste Boxes (SWB) and Ten Drum Overpacks (TDOP), for subsequent characterization and WIPP disposal, commenced in FY04. To date, 10 Black Boxes have been repackaged, resulting in 40 SLB-2's, and 37 B25 overpack boxes, these B25's will be overpacked in SLB-2's prior to shipping to WIPP. This paper will describe experience to date from this project.

  5. Modeling the circulation of the Dead Sea

    NASA Astrophysics Data System (ADS)

    Brenner, Steve; Lensky, Nadav; Gertman, Isaac

    2015-04-01

    The Dead Sea is a hypersaline, terminal lake located at the lowest point on the land surface of the Earth. Its current level is more than 429 m below MSL, and due to a negative water balance (mainly anthropogenic), the lake level has been dropping at an average rate of more than 1 m/yr for more than 30 years. The mean salinity has also been steadily increasing and today is close to 280 psu. The region of the Dead Sea is a unique landscape that has important historical, cultural, and economic value and therefore such an extreme change of the lake has significant environmental and economic consequences. In recent years there has been a notable increase in observing and monitoring of the lake through continuous measurements from several fixed buoys as well as during quasi-regular cruises. In order to complement the measurements and improve our understanding of the dynamics of this unique lake a three dimensional circulation model is being developed. Previous modeling efforts were limited mainly to a one dimensional column model which was coupled to a comprehensive physio-chemical model and used for long term multi-decadal simulations. In this study the focus is on understanding the dynamical processes that control the lake-wide circulation on time scales ranging from days to seasons. The first step was to replace the equation of state with an equation appropriate for the hypersaline conditions, in addition to some minor tuning of the turbulence closure scheme. Results will be presented from preliminary simulations of the wind driven circulation in various seasons. A case study of a recent unusual winter flooding event, during which the lake level rose by more than 20 cm over a two month period, will also be presented. The model successfully simulated the observed transition from holomictic to meromictic conditions and epilimnion dilution during this event, as well as the restoration of holomictic conditions when the level started to drop again. The relationship

  6. Micro-facies of Dead Sea sediments

    NASA Astrophysics Data System (ADS)

    Neugebauer, Ina; Schwab, Markus J.; Brauer, Achim; Frank, Ute; Dulski, Peter; Kitagawa, Hiroyuki; Enzel, Yehouda; Waldmann, Nicolas; Ariztegui, Daniel; Drilling Party, Dsddp

    2013-04-01

    Lacustrine sediments infilling the Dead Sea basin (DSB) provide a rare opportunity to trace changing climates in the eastern Mediterranean-Levant region throughout the Pleistocene and Holocene. In this context, high-resolution investigation of changes in sediment micro- facies allow deciphering short-term climatic fluctuations and changing environmental conditions in the Levant. The Dead Sea is a terminal lake with one of the largest drainage areas in the Levant, located in the Mediterranean climate zone and influenced also by the Saharo-Arabian deserts. Due to drastic climatic changes in this region, an exceptionally large variety of lacustrine sediments has been deposited in the DSB. These sediments, partially the results of changing lake levels, primarily represent changes in precipitation (e.g. Enzel et al., 2008). Evaporites (halite and gypsum) reflect dry climatic conditions during interglacials, while alternated aragonite-detritus (AAD) is deposited during glacial lake level high-stands. Here we present the first micro-facies inventory of a ~450 m long sediment profile from the deepest part of the northern DSB (ICDP site 5017-1, ~300 m water depth). The sediment record comprises the last two glacial-interglacial cycles, with mainly AAD facies in the upper part of the Amora Formation (penultimate glacial) and the last glacial Lisan Formation. The last interglacial Samra and the Holocene Zeelim Formations are predominantly characterized by thick bedded halite deposits, intercalated by partly laminated detrital marl sequences. Representative sections of the different facies types have been analyzed for micro-facies on petrographic thin sections, supported by high-resolution µXRF element scanning, magnetic susceptibility measurements and microscopic fluorescence analysis. Furthermore, Holocene sediments retrieved at the deep basin core site have been compared to their shallow-water counterpart at the western margin of the lake (core DSEn; Migowski et al., 2004

  7. Origin of Self-Replicating Biopolymers: Autocatalytic Feedback Can Jump-Start the RNA World

    NASA Astrophysics Data System (ADS)

    Wu, M.; Higgs, P. G.

    2010-04-01

    We study a chemical reaction model of RNA polymerization. The system undergoes a transition from a dead state with negligible ribozyme concentration to a living state where ribozyme concentration is high and synthesis of ribozymes is autocatalytic.

  8. Dead Time of Single Photon Avalanche Diodes

    NASA Astrophysics Data System (ADS)

    Neri, L.; Tudisco, S.; Musumeci, F.; Scordino, A.; Fallica, G.; Mazzillo, M.; Zimbone, M.

    2011-06-01

    Single Photon Avalanche Diode (SPAD) is the new generation of Geiger-Muller counter device developed in semiconductor technology [S. Privitera et al. Sensors Journal, vol 8 Iss. 8 (2008) 4636; S. Tudisco et al. IEEE Sensors Journal vol 8 ISS 7-8 (2008) 1324; S. Cova et al. Applied Optics 35 (1996) 1956]. Physical dead time model and noise production process has been analyzed and their corrections have been performed [S.H. Lee, R.P. Gardner, M. Jae, Nucl. Instr. and Meth. in Phys. Res. B 263 (2007) 46]. We have been able to extract the real amount of incident photon rate up to 10 7cps using a device with 0.97μs total deadtime. We also developed the equation of the noise count rate vs incoming photon rate, supported by Montecarlo simulation and experimental data. We marked the difference between dark rate and noise count rate, and introduced the noise rate inside the hybrid deadtime equation used for SPAD device.

  9. Is Space-based Interferometry Dead?

    NASA Astrophysics Data System (ADS)

    Leisawitz, David; Benford, D.; Blain, A.; Carr, J.; Fich, M.; Fischer, J.; Goldsmith, P.; Greaves, J.; Griffin, M.; Helou, G.; Ivison, R.; Kuchner, M.; Lyon, R.; Matsuo, H.; Rinehart, S. A.; Serabyn, E.; Shibai, H.; Silverberg, R.; Staguhn, J.; Unwin, S.; Wilner, D.; Wootten, A.; Wright, E. L.

    2011-05-01

    In the wake of the Decadal Survey and a January 2011 meeting of NASA's Exoplanet Exploration Program Analysis Group (ExoPAG), one might be tempted to conclude that space interferometry is dead. We explain why this slogan is hyperbole, summarize the steps currently being taken to prepare for a space-based far-IR interferometer, and reiterate the science case for an imaging and spectroscopic interferometer - SPIRIT - that would operate in space at long infrared wavelengths. Space-based interferometry is alive and well, but the center of activity has shifted to a spectral region (25 to 400 microns) in which no alternative measurement technique can provide information essential to answering several scientific questions deemed compelling by the Decadal Survey. Astrophysicists will use SPIRIT to: discover how the conditions for habitability arise during planetary system formation; find and characterize exoplanets by measuring their sculpting effects on protoplanetary and debris disks; and study the formation, merger history, and star formation history of galaxies.

  10. Dead Reckoning Pedometer Graphical User Interface

    2003-04-26

    The Dead Reckoning Pedometer Graphical User Interface (DRP GUI) software is tasked with maturing the technology described in a WSRC patent application. This patent application describes an electronic navigation system that records human foot movements, in three dimensions, for the purpose of determining position, distance, and speed of a walking person. The simiplest form of the apparatus consists of a magnetometer (an instrument that measures magnetic field strength) on one foot and a small permanentmore » magnet on another foot with pressure sensors on each foot. When a person takes a step, the foot will hit the ground and produce a signal on the pressure sensor. This will trigger a reading of the magnetometer so that the relative position of one foot to the other can be calculated. This same process is repeated for each step. The DRP could be very useful for tracking emergency personnel such as firemen, policemen, and paramedics when they travel within a building. Technologies such as global positioning systems to not work within buildings. The goal of the DRP GUI V1.0.0 software is to provide a three-dimensional graphical user interface that will allow WSRC to demonstrate the DRP concepts to potential patent licensees. It is hoped that a partnership will allow WSRC and another company to further develop the DRP technology and software into a commercial product.« less

  11. Dead Reckoning Pedometer Graphical User Interface

    SciTech Connect

    Harpring, Larry

    2003-04-26

    The Dead Reckoning Pedometer Graphical User Interface (DRP GUI) software is tasked with maturing the technology described in a WSRC patent application. This patent application describes an electronic navigation system that records human foot movements, in three dimensions, for the purpose of determining position, distance, and speed of a walking person. The simiplest form of the apparatus consists of a magnetometer (an instrument that measures magnetic field strength) on one foot and a small permanent magnet on another foot with pressure sensors on each foot. When a person takes a step, the foot will hit the ground and produce a signal on the pressure sensor. This will trigger a reading of the magnetometer so that the relative position of one foot to the other can be calculated. This same process is repeated for each step. The DRP could be very useful for tracking emergency personnel such as firemen, policemen, and paramedics when they travel within a building. Technologies such as global positioning systems to not work within buildings. The goal of the DRP GUI V1.0.0 software is to provide a three-dimensional graphical user interface that will allow WSRC to demonstrate the DRP concepts to potential patent licensees. It is hoped that a partnership will allow WSRC and another company to further develop the DRP technology and software into a commercial product.

  12. The spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of the eukaryotic box C/D snoRNP nucleotide modification complex.

    PubMed

    Qu, Guosheng; van Nues, Rob W; Watkins, Nicholas J; Maxwell, E Stuart

    2011-01-01

    Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.

  13. Dead pixel correction techniques for dual-band infrared imagery

    NASA Astrophysics Data System (ADS)

    Nguyen, Chuong T.; Mould, Nick; Regens, James L.

    2015-07-01

    We present two new dead pixel correction algorithms for dual-band infrared imagery. Specifically, we address the problem of repairing unresponsive elements in the sensor array using signal processing techniques to overcome deficiencies in image quality that are present following the nonuniformity correction process. Traditionally, dead pixel correction has been performed almost exclusively using variations of the nearest neighbor technique, where the value of the dead pixel is estimated based on pixel values associated with the neighboring image structure. Our approach differs from existing techniques, for the first time we estimate the values of dead pixels using information from both thermal bands collaboratively. The proposed dual-band statistical lookup (DSL) and dual-band inpainting (DIP) algorithms use intensity and local gradient information to estimate the values of dead pixels based on the values of unaffected pixels in the supplementary infrared band. The DSL algorithm is a regression technique that uses the image intensities from the reference band to estimate the dead pixel values in the band undergoing correction. The DIP algorithm is an energy minimization technique that uses the local image gradient from the reference band and the boundary values from the affected band to estimate the dead pixel values. We evaluate the effectiveness of the proposed algorithms with 50 dual-band videos. Simulation results indicate that the proposed techniques achieve perceptually and quantitatively superior results compared to existing methods.

  14. Dead space mask eliminates central apnea at altitude.

    PubMed

    Patz, David S; Patz, Michael D; Hackett, Peter H

    2013-06-01

    Travelers to high altitude may have disturbed sleep due to periodic breathing with frequent central apneas. We tested whether a mask with added dead space could reduce the central apneas of altitude. 16 subjects were recruited, age 18-35, residing at 4600 ft (1400 m). They each slept one night with full polysomnographic monitoring, including end tidal CO2, in a normobaric hypoxia tent simulating 12,000 ft. (3658 m) altitude. Those who had a central apnea index (CAI) >20/h returned for a night in the tent for dead space titration, during which they slept with increasing amounts of dead space, aiming for a CAI <5/h or <10% of baseline. Then each subject slept another night with the titrated amount of dead space. Of the 16 subjects, 5 had a central apnea index >20/h mean 49.1, range 21.4-131.5/hr. In each of the 5, the dead space mask reduced the CAI by at least 88% to a mean of 3.1, range 0.9-7.1/h, (p=0.04). Hypopnea index was unchanged. Three subjects required 500 cc of dead space or less. One subject required 860 cc, and one required 2.1 L. Morning symptoms and arousal index were not significantly affected by the dead space mask. Dead space did not appear to increase the CO2 reserve. At 12,000 ft., central apneas can be effectively reduced with a dead space mask, but clinical utility will require further evaluation.

  15. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  16. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  17. 36 CFR 1192.33 - Fare box.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 36 Parks, Forests, and Public Property 3 2013-07-01 2012-07-01 true Fare box. 1192.33 Section 1192.33 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD... Systems § 1192.33 Fare box. Where provided, the farebox shall be located as far forward as practicable...

  18. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  19. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  20. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  1. BLS: Box-fitting Least Squares

    NASA Astrophysics Data System (ADS)

    Kovács, G.; Zucker, S.; Mazeh, T.

    2016-07-01

    BLS (Box-fitting Least Squares) is a box-fitting algorithm that analyzes stellar photometric time series to search for periodic transits of extrasolar planets. It searches for signals characterized by a periodic alternation between two discrete levels, with much less time spent at the lower level.

  2. Cereal Box Design: An Interdisciplinary Graphics Activity

    ERIC Educational Resources Information Center

    Fitzgerald, Mike; Tsosie, Teri

    2004-01-01

    This article describes cereal box design, an interdisciplinary graphics activity. The cereal box design activity is intriguing both for its simplicity and the resourcefulness that it can generate in young people. It lends itself to a variety of curriculums. It covers both consumerism and Design for the Environment (DfE) concepts broadly and in…

  3. 49 CFR 38.33 - Fare box.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Fare box. 38.33 Section 38.33 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 38.33 Fare box. Where provided, the...

  4. Box Plots in the Australian Curriculum

    ERIC Educational Resources Information Center

    Watson, Jane M.

    2012-01-01

    This article compares the definition of "box plot" as used in the "Australian Curriculum: Mathematics" with other definitions used in the education community; describes the difficulties students experience when dealing with box plots; and discusses the elaboration that is necessary to enable teachers to develop the knowledge necessary to use them…

  5. Cosmetic Foot Surgery: Fashion's Pandora's Box

    MedlinePlus

    ... Fashion’s Pandora’s Box? A A A | Print | Share Cosmetic Foot Surgery: Fashion’s Pandora’s Box? Foot and ankle ... extreme and imprudent as it may sound, the cosmetic surgery craze isn't just for faces anymore- ...

  6. Structural Basis for Substrate Placement by an Archaeal Box C/D Ribonucleoprotein Particle

    SciTech Connect

    Xue, Song; Wang, Ruiying; Yang, Fangping; Terns, Rebecca M.; Terns, Michael P.; Zhang, Xinxin; Maxwell, E.Stuart; Li, Hong

    2012-05-09

    Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs.

  7. 46 CFR 111.81-3 - Cables entering boxes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Cables entering boxes. 111.81-3 Section 111.81-3...-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-3 Cables entering boxes. Each cable entering a box or fitting must be protected from abrasion and must meet the following: (a) Each...

  8. 46 CFR 111.81-3 - Cables entering boxes.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Cables entering boxes. 111.81-3 Section 111.81-3...-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-3 Cables entering boxes. Each cable entering a box or fitting must be protected from abrasion and must meet the following: (a) Each...

  9. 46 CFR 111.81-3 - Cables entering boxes.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Cables entering boxes. 111.81-3 Section 111.81-3...-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-3 Cables entering boxes. Each cable entering a box or fitting must be protected from abrasion and must meet the following: (a) Each...

  10. 46 CFR 111.81-3 - Cables entering boxes.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Cables entering boxes. 111.81-3 Section 111.81-3...-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-3 Cables entering boxes. Each cable entering a box or fitting must be protected from abrasion and must meet the following: (a) Each...

  11. 46 CFR 111.81-3 - Cables entering boxes.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Cables entering boxes. 111.81-3 Section 111.81-3...-GENERAL REQUIREMENTS Outlet Boxes and Junction Boxes § 111.81-3 Cables entering boxes. Each cable entering... through which a conductor enters must be closed. (b) Cable armor must be secured to the box or fitting....

  12. Uncertainty evaluation of dead zone of diagnostic ultrasound equipment

    NASA Astrophysics Data System (ADS)

    Souza, R. M.; Alvarenga, A. V.; Braz, D. S.; Petrella, L. I.; Costa-Felix, R. P. B.

    2016-07-01

    This paper presents a model for evaluating measurement uncertainty of a feature used in the assessment of ultrasound images: dead zone. The dead zone was measured by two technicians of the INMETRO's Laboratory of Ultrasound using a phantom and following the standard IEC/TS 61390. The uncertainty model was proposed based on the Guide to the Expression of Uncertainty in Measurement. For the tested equipment, results indicate a dead zone of 1.01 mm, and based on the proposed model, the expanded uncertainty was 0.17 mm. The proposed uncertainty model contributes as a novel way for metrological evaluation of diagnostic imaging by ultrasound.

  13. Spreading dead zones and consequences for marine ecosystems.

    PubMed

    Diaz, Robert J; Rosenberg, Rutger

    2008-08-15

    Dead zones in the coastal oceans have spread exponentially since the 1960s and have serious consequences for ecosystem functioning. The formation of dead zones has been exacerbated by the increase in primary production and consequent worldwide coastal eutrophication fueled by riverine runoff of fertilizers and the burning of fossil fuels. Enhanced primary production results in an accumulation of particulate organic matter, which encourages microbial activity and the consumption of dissolved oxygen in bottom waters. Dead zones have now been reported from more than 400 systems, affecting a total area of more than 245,000 square kilometers, and are probably a key stressor on marine ecosystems.

  14. Adaptive control with variable dead-zone nonlinearities

    NASA Technical Reports Server (NTRS)

    Orlicki, D.; Valavani, L.; Athans, M.; Stein, G.

    1984-01-01

    It has been found that fixed error dead-zones as defined in the existing literature result in serious degradation of performance, due to the conservativeness which characterizes the determination of their width. In the present paper, variable width dead-zones are derived for the adaptive control of plants with unmodeled dynamics. The derivation makes use of information available about the unmodeled dynamics both a priori as well as during the adaptation process, so as to stabilize the adaptive loop and at the same time overcome the conservativeness and performance limitations of fixed-dead zone adaptive or fixed gain controllers.

  15. Conserved Composition of Mammalian Box H/ACA and Box C/D Small Nucleolar Ribonucleoprotein Particles and Their Interaction with the Common Factor Nopp140

    PubMed Central

    Yang, Yunfeng; Isaac, Cynthia; Wang, Chen; Dragon, François; Pogac̆ić, Vanda; Meier, U. Thomas

    2000-01-01

    Small nucleolar ribonucleoprotein particles (snoRNPs) mainly catalyze the modification of rRNA. The two major classes of snoRNPs, box H/ACA and box C/D, function in the pseudouridylation and 2′-O-methylation, respectively, of specific nucleotides. The emerging view based on studies in yeast is that each class of snoRNPs is composed of a unique set of proteins. Here we present a characterization of mammalian snoRNPs. We show that the previously characterized NAP57 is specific for box H/ACA snoRNPs, whereas the newly identified NAP65, the rat homologue of yeast Nop5/58p, is a component of the box C/D class. Using coimmunoprecipitation experiments, we show that the nucleolar and coiled-body protein Nopp140 interacts with both classes of snoRNPs. This interaction is corroborated in vivo by the exclusive depletion of snoRNP proteins from nucleoli in cells transfected with a dominant negative Nopp140 construct. Interestingly, RNA polymerase I transcription is arrested in nucleoli depleted of snoRNPs, raising the possibility of a feedback mechanism between rRNA modification and transcription. Moreover, the Nopp140-snoRNP interaction appears to be conserved in yeast, because depletion of Srp40p, the yeast Nopp140 homologue, in a conditional lethal strain induces the loss of box H/ACA small nucleolar RNAs. We propose that Nopp140 functions as a chaperone of snoRNPs in yeast and vertebrate cells. PMID:10679015

  16. Casticin induces breast cancer cell apoptosis by inhibiting the expression of forkhead box protein M1

    PubMed Central

    LIU, LI-PING; CAO, XIAO-CHENG; LIU, FEI; QUAN, MEI-FANG; SHENG, XI-FENG; REN, KAI-QUN

    2014-01-01

    Casticin is an active ingredient derived from Fructus Viticis, a traditional Chinese medicine. This study aimed to investigate the role of forkhead box O3 (FOXO3a) in breast cancer cells and examine the regulatory mechanisms of FOXO3a in response to casticin treatment of the cells by ELISA, flow cytometry, small interfering RNA (siRNA) transfection and western blot analysis. Casticin treatment induced apoptosis and reduced the expression of the transcription factor forkhead box protein M1 (FOXM1). In addition, FOXM1 repression induced by casticin treatment was associated with the activation of FOXO3a via increased dephosphorylation. Notably, silencing FOXO3a expression by siRNA-mediated gene knockdown attenuated casticin-mediated apoptosis. Collectively, these findings suggest that FOXO3a is a critical mediator of the inhibitory effects of casticin on apoptosis in breast cancer cells. PMID:24765206

  17. The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs.

    PubMed Central

    Winkler, W C; Grundy, F J; Murphy, B A; Henkin, T M

    2001-01-01

    Two different transcription termination control mechanisms, the T box and S box systems, are used to regulate transcription of many bacterial aminoacyl-tRNA synthetase, amino acid biosynthesis, and amino acid transport genes. Both of these regulatory mechanisms involve an untranslated mRNA leader region capable of adopting alternate structural conformations that result in transcription termination or transcription elongation into the downstream region. Comparative analyses revealed a small RNA secondary structural element, designated the GA motif, that is highly conserved in both T box and S box leader sequences. The motif consists of two short helices separated by an asymmetric internal loop, with highly conserved GA dinucleotide sequences on either side of the internal loop. Site-directed mutagenesis of this motif in model T and S box leader sequences indicated that it is essential for transcriptional regulation in both systems. This motif is similar to the binding site of yeast ribosomal protein L30, the Snu13p binding sites found in U4 snRNA and box C/D snoRNAs, and two elements in 23S rRNA. PMID:11497434

  18. North American box turtles: A natural history

    USGS Publications Warehouse

    Dodd, C. Kenneth

    2002-01-01

    Once a familiar backyard visitor in many parts of the United States and Mexico, the box turtle is losing the battle against extinction. In North American Box Turtles, C. Kenneth Dodd, Jr., has written the first book-length natural history of the twelve species and subspecies of this endangered animal. This volume includes comprehensive information on the species’ evolution, behavior, courtship and reproduction, habitat use, diet, population structure, systematics, and disease. Special features include color photos of all species, subspecies, and their habitats; a simple identification guide to both living and fossil species; and a summary of information on fossil Terrapene and Native uses of box turtles. End-of-chapter sections highlight future research directions, including the need for long-term monitoring and observation of box turtles within their natural habitat and conservation applications. A glossary and a bibliography of literature on box turtles accompany the text.

  19. Groundwater-Lake Interaction in the Dead Sea Area

    NASA Astrophysics Data System (ADS)

    Kiro, Y.; Weinstein, Y.; Starinsky, A.; Yechieli, Y.

    2011-12-01

    The Dead Sea hypersaline water system is unique in terms of its unusual geochemical composition, rapid lake level changes and water composition of the brines discharging along its shoreline. The Dead Sea can be used as a natural lab for studying groundwater-seawater interaction and saline water hydrological circulation along the aquifer-sea boundary. It provides an opportunity to follow the geochemical processes along a flow path from the lake into the aquifer and back into the lake. The lake level has been dropping since the 1960's due to human interference in its water budget, reaching a rate of 1 m/yr in recent years. Saline water circulation in coastal aquifers may be a major process that governs trace element mass balances in coastal areas. This study uses radium isotopes in order to quantify the lake water circulation in the Dead Sea aquifer. There are four naturally-occurring radium isotopes, with half-lives ranging from 3.7 days to 1600 years which are chain products of uranium and thorium isotopes. Radium isotopes are usually enriched in saline groundwater and therefore are good candidates for estimating seawater or hypersaline lake water circulation in the aquifer. Compared to most natural water bodies, the Dead Sea is extremely enriched in radium and barium, where both 226Ra and 228Ra activities and Ba concentration (145, 1-2 dpm/L and 5 mg/L, respectively) are 2-3 orders of magnitude higher than in ocean water, whereas the salinity of the Dead Sea is only 10 times higher. Circulated Dead Sea water in the aquifer contains decreased concentrations of 226Ra (60 dpm/L), Ba (1.5 mg/L), Sr (300 relative to 340 mg/L in the Dead Sea) and Sulfate (250 relative to 392 mg/L). We suggest that the low 226Ra and Ba concentrations are due to precipitation of barite and celestine from the supersaturated Dead Sea water on entering the aquifer. 228Ra and the shorter-lived 224Ra and 223Ra, which have much lower activities in the Dead Sea (up to 1.8, 3 and 0.8 dpm

  20. Cirrhosis and trauma: a deadly duo.

    PubMed

    Christmas, A Britton; Wilson, Ashley K; Franklin, Glen A; Miller, Frank B; Richardson, J David; Rodriguez, Jorge L

    2005-12-01

    It has been previously reported that trauma patients with cirrhosis undergoing emergency abdominal operations exhibit a fourfold increase in mortality independent of their Child's classification. We undertook this review to assess the impact of cirrhosis on trauma patients. We reviewed the records of patients from 1993 to 2003 with documented hepatic cirrhosis and compared them to a 2:1 control population without hepatic cirrhosis and matched for age, sex, Injury Severity Score (ISS), and Glasgow Coma Score (GCS). Demographic, severity of injury, and outcome data were recorded. Student's t test and X2 were used for statistical analysis and a P < 0.05 was significant. Sixty-one patients had documented cirrhosis and were compared to 156 matched controls. Comparing the two groups demonstrates there was no difference in age, ISS, or GCS. Intensive care stay, hospital length of stay, blood requirements in the first 24 hours postinjury, and mortality (33% vs 1%) was significantly greater in the trauma patients with cirrhosis. Fifty-five per cent of deaths in the cirrhosis group was due to sepsis, and, as the Child's class increases, so does the mortality (Child's A, 15%; B, 37%; and C, 63%). In 64 per cent of cirrhotics without an emergent abdominal operation, mortality was 21 per cent. In the 36 per cent of cirrhotics who had emergent abdominal operation, mortality was 55 per cent. Hepatic cirrhosis in trauma patients, regardless of severity of injury or the need for an abdominal intervention, is a poor prognostic indicator. The necessity of an abdominal operative intervention further amplifies this effect. Trauma and cirrhosis is, in fact, a deadly duo.

  1. Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes.

    PubMed

    Tanabe, Yoichi; Hasebe, Mitsuyasu; Sekimoto, Hiroyuki; Nishiyama, Tomoaki; Kitani, Masakazu; Henschel, Katrin; Münster, Thomas; Theissen, Günter; Nozaki, Hisayoshi; Ito, Motomi

    2005-02-15

    The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.

  2. [Prognosis of affinity change of the TATA-binding protein to TATA-boxes upon polymorphisms of the human gene promoter TATA boxes].

    PubMed

    Ponomarenko, P M; Ponomarenko, M P; Drachkova, I A; Lysova, M V; Arshinova, T V; Savinkova, L K; Kolchanov, N A

    2009-01-01

    TATA-binding protein (TBP) is a subunit of basal transcription factor TFIID that recognizes and binds to the TATA-box on TATA-containing promoters of class II genes, and starts assembling RNA polymerase II basal transcription complex. It is shown in many works that the sequence of TATA-box with its flanking regions affects the level of basal and activated transcription. TATA-box polymorphisms and human hereditary diseases associated with them show that TBP/TATA interaction may indirectly affect gene regulation in vivo. The object of this work is to determine changes in the TBP/TATA affinity upon polymorphisms in TATA-boxes of human gene promoters. We assess changes in TBP/TATA affinities in silico by using our formula of equilibrium TBP/TATA binding upon four consecutive steps: nonspecific binding <--> sliding <--> braking (stopping) <--> stabilization. Our prognoses agree with known examples of TATA-box polymorphisms and human hereditary diseases associated with them. PMID:19548537

  3. Raising the Dead without a Red Sea-Dead Sea Canal? A hydro-economic-institutional analysis

    NASA Astrophysics Data System (ADS)

    Rosenberg, D. E.

    2010-12-01

    Presently, just 100 million cubic meters per year (MCM/year) of the 1,000+ MCM/year that historically flowed in the lower Jordan River reach the Dead Sea. Israeli, Jordanian, and Syrian dam and extraction projects built over seven decades have principally caused the reduced flow, associated falling Dead Sea level, shrinking surface area, sink holes, salinity, and other catastrophic problems. These problems will be magnified in the face of up to 20% reductions in precipitation expected with climate change. The fix proposed by Jordan, Israel, and Palestine—and now under study by the World Bank—envisions building a $US 5 billion multipurpose canal from the Red Sea to the Dead Sea that would also generate hydropower and desalinated water. Yet alternatives to raise the Dead Sea level that could take advantage of hydrologic variability remain unstudied. Here we show system-wide hydrologic and economic impacts of and discusses institutional management for alternatives to raise the Dead Sea level. Hydro-economic model results for the inter-tied Israel-Jordan-Palestinian water systems show the desalination component of the Red Sea-Dead Sea project is economically unviable. Further, many decentralized new supply, wastewater reuse, conveyance, conservation, and leak reduction projects and programs in each country together increase economic benefits and can reliably deliver up to 900 MCM/year to the Dead Sea. In all cases, results show that net benefits fall and water scarcity rises as the flow volume delivered to the Dead Sea increases. These findings suggest that (i) each country has little individual incentive to allow water to flow to the Dead Sea, and (ii) outside institutions—such as the World Bank—that seek to raise the Dead should instead offer the countries direct incentives to deliver water rather than build them new infrastructure. The work expands the set of viable options to raise the Dead Sea level and can help the World Bank and others recommend whether

  4. Genome-wide analyses of retrogenes derived from the human box H/ACA snoRNAs

    PubMed Central

    Li, Yuping Luo and Siguang

    2007-01-01

    The family of box H/ACA snoRNA is an abundant class of non-protein-coding RNAs, which play important roles in the post-transcriptional modification of rRNAs and snRNAs. Here we report the characterization in the human genome of 202 sequences derived from box H/ACA snoRNAs. Most of them were retrogenes formed using the L1 integration machinery. About 96% of the box H/ACA RNA-related sequences are found in corresponding locations on the chimpanzee and human chromosomes, while the mouse shares ∼50% of these human sequences, suggesting that some of the H/ACA RNA-related sequences in primate occurred after the rodent/primate divergence. Of the H/ACA RNA-related sequences, 49% are found in intronic regions of protein-coding genes and 64 H/ACA-related sequences can be folded to the typical secondary structure of the box H/ACA snoRNA family, while 30 of them were recognized as functional homologs of their corresponding box H/ACA snoRNAs previously reported. Of the 64 sequences with the typical secondary structure of the box H/ACA RNA family, 11 were found in EST databases and 5 among which were shown to be expressed in more than one human tissue. Notably, U107f is nested in an intron of a protein gene coding for nudix-type motif 13, but expressed from the opposite strand, and the searching of EST databases revealed it can be expressed in liver and spleen, even in melanotic melanoma. PMID:17175533

  5. Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

    PubMed Central

    Talkish, Jason; Zhang, Jingyu; Jakovljevic, Jelena; Horsey, Edward W.; Woolford, John L.

    2012-01-01

    To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the ‘B-factor’ proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes. PMID:22735702

  6. Assessment and Management of Dead-Wood Habitat

    USGS Publications Warehouse

    Hagar, Joan

    2007-01-01

    Introduction The Bureau of Land Management (BLM) is in the process of revising its resource management plans for six districts in western and southern Oregon as the result of the settlement of a lawsuit brought by the American Forest Resource Council. A range of management alternatives is being considered and evaluated including at least one that will minimize reserves on O&C lands. In order to develop the bases for evaluating management alternatives, the agency needs to derive a reasonable range of objectives for key issues and resources. Dead-wood habitat for wildlife has been identified as a key resource for which decision-making tools and techniques need to be refined and clarified. Under the Northwest Forest Plan, reserves were to play an important role in providing habitat for species associated with dead wood (U.S. Department of Agriculture Forest Service and U.S. Department of the Interior Bureau of Land Management, 1994). Thus, the BLM needs to: 1) address the question of how dead wood will be provided if reserves are not included as a management strategy in the revised Resource Management Plan, and 2) be able to evaluate the effects of alternative land management approaches. Dead wood has become an increasingly important conservation issue in managed forests, as awareness of its function in providing wildlife habitat and in basic ecological processes has dramatically increased over the last several decades (Laudenslayer et al., 2002). A major concern of forest managers is providing dead wood habitat for terrestrial wildlife. Wildlife in Pacific Northwest forests have evolved with disturbances that create large amounts of dead wood; so, it is not surprising that many species are closely associated with standing (snags) or down, dead wood. In general, the occurrence or abundance of one-quarter to one-third of forest-dwelling vertebrate wildlife species, is strongly associated with availability of suitable dead-wood habitat (Bunnell et al., 1999; Rose et al

  7. Smart City Planning Can Cut Deadly Diseases, Improve Air Quality

    MedlinePlus

    ... https://medlineplus.gov/news/fullstory_161133.html Smart City Planning Can Cut Deadly Diseases, Improve Air Quality Study ... three quarters of this population living in cities, city planning must be part of a comprehensive solution to ...

  8. Extreme flood events in the Dead Sea basin

    NASA Astrophysics Data System (ADS)

    Ahlborn, Marieke; Ben Dor, Yoav; Schwab, Markus J.; Neugebauer, Ina; Plessen, Birgit; Tjallingii, Rik; Erel, Yigal; Enzel, Yehouda; Brauer, Achim

    2016-04-01

    The Dead Sea is a hypersaline, terminal lake located within the Dead Sea basin at the lowest continental elevation on Earth (~425 m below mean sea level). Extreme hydro-meteorological events in terms of flash floods occur regularly during the wet season in the Dead Sea basin and adjacent mountain ranges. However, little is known about the impact of these extreme floods on the sedimentary dynamics in the Dead Sea and possible links to long-term climate changes. The trilateral research project PALEX (Paleoclimate in the Eastern Mediterranean Region - Levante: Paleohydrology and Extreme Flood Events) was recently initiated within the framework of the DFG priority program 1006 ICDP (International Continental Scientific Drilling Program) to investigate extreme flood events in the Dead Sea basin during the Late Pleistocene and Holocene. Within the ICDP Dead Sea Deep Drilling Project (DSDDP) the ~455 m long sediment core 5017-1 was recovered from the northern Dead Sea basin. Previously published results (Neugebauer et al., 2014, 2015) have demonstrated the occurrence of extreme flood events represented in the sediments as thick graded detrital layers during Late Holocene dry phases. Based on these results we will apply a comprehensive analytical approach including microfacies analyses, μXRF element scanning, and stable isotope geochemistry to different time intervals of core 5017-1. Particularly, we aim to investigate the structure and composition of detrital layers in order to decipher sediment transport mechanisms and the provenance of the flood-triggered sediments. The overarching goal is to establish a high-resolution extreme flood time series for the Dead Sea basin on the basis of a previously established radiocarbon and U-Th chronology (Torfstein et al., 2015; Neugebauer et al., 2014) and to study a possible link between the frequency and magnitude of extreme flood events and the long-term climate trend. Neugebauer I, Brauer A, Schwab MJ, et al. (2014) Lithology of

  9. An inverted TATA box directs downstream transcription of the bone sialoprotein gene.

    PubMed Central

    Li, J J; Kim, R H; Sodek, J

    1995-01-01

    The orientation of the TATA box is thought to direct downstream transcription of eukaryotic genes by RNA polymerase II. However, the putative TATA box in the promoter of the bone sialoprotein (BSP) gene, which codes for a tissue-specific and developmentally regulated bone matrix protein, is inverted (5'-TTTATA-3') relative to the consensus TATA box sequence (5'-TATAAA-3') and is overlapped by a vitamin D3-response element. Here we show that the inverted TATA sequence in the rat BSP gene binds to recombinant TATA-box-binding protein (TBP) with an affinity similar to that observed with the consensus TATA box, and site-directed point mutations in the inverted TATA sequence (mutating TTTATA into TCTCTA) abrogate both TBP binding and BSP promoter activity. However, when the inverted TATA sequence is changed to a canonical TATAAA, the TBP- and vitamin D3 receptor-binding properties together with the BSP promoter activity are retained. In addition, we found that the TBP is required to reconstitute in vitro transcription driven by the BSP promoter. These studies, which have revealed a naturally occurring inverted TATA box that can bind TBP and direct downstream transcription, demonstrate that the orientation of the TATA box does not determine the direction of transcription in higher eukaryotic genes. Consequently, the inverted TATA box that is conserved in the human, rat and mouse BSP gene promoters will provide an excellent in vivo model to investigate the polarity of the transcription factor IID-DNA complex and its relation to downstream transcription. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 PMID:7646464

  10. 6. VIEW OF SPILLWAY TIMBERS AND WATER CONTROL BOX, SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. VIEW OF SPILLWAY TIMBERS AND WATER CONTROL BOX, SHOWING WATER CONTROL BOX WITH LOWERED LAKE LEVEL - Three Bears Lake & Dams, Water Control Box, North of Marias Pass, East Glacier Park, Glacier County, MT

  11. Experimental dead-time distortions of poisson processes

    NASA Astrophysics Data System (ADS)

    Faraci, G.; Pennisi, A. R.

    1983-07-01

    In order to check the distortions, introduced by a non-extended dead time on the Poisson statistics, accurate experiments have been made in single channel counting. At a given measuring time, the dependence on the choice of the time origin and on the width of the dead time has been verified. An excellent agreement has been found between the theoretical expressions and the experimental curves.

  12. Teleseismic traveltimes residuals across the Dead Sea basin

    NASA Astrophysics Data System (ADS)

    Hofstetter, A.; Dorbath, C.

    2014-12-01

    New findings of the structure of the Dead Sea sedimentary basin and its eastern and western bordering regions are obtained by P and PKP wave relative traveltime residuals of 644 teleseisms, as recorded by the Dead Sea Integrated Research portable seismic network in the Dead Sea basin and its neighboring regions. The Lisan Peninsula is characterized by relatively small teleseismic traveltime residuals of about 0.14 s, in the latitude range of 31.22°-31.37° and at the longitude of 35.50°, slowly decreasing toward the west. The largest teleseismic traveltime residuals are in the southern Dead Sea basin, south of the Lisan Peninsula in the latitude range of 31.05°-31.15° and along longitude 35.45° and continuing southward toward the Amaziahu Fault, reaching values of 0.4-0.5 s. There is a small positive residual at the Amaziahu Fault and a small negative residual south of it probably marking the southern end of the Dead Sea basin. East and west of the Dead Sea basin the mean teleseismic traveltime residuals are negative with overall averages of -0.35 s and -0.45 s, respectively. Using the teleseismic residuals, we estimate the horizontal dimensions of the Lisan salt diapir to be 23 km × 13 km at its widest and a maximal thickness of about 7.2 km. The thickness of the Mount Sodom salt diapir is estimated as 6.2 km.

  13. The dead donor rule, voluntary active euthanasia, and capital punishment.

    PubMed

    Coons, Christian; Levin, Noah

    2011-06-01

    We argue that the dead donor rule, which states that multiple vital organs should only be taken from dead patients, is justified neither in principle nor in practice. We use a thought experiment and a guiding assumption in the literature about the justification of moral principles to undermine the theoretical justification for the rule. We then offer two real world analogues to this thought experiment, voluntary active euthanasia and capital punishment, and argue that the moral permissibility of terminating any patient through the removal of vital organs cannot turn on whether or not the practice violates the dead donor rule. Next, we consider practical justifications for the dead donor rule. Specifically, we consider whether there are compelling reasons to promulgate the rule even though its corresponding moral principle is not theoretically justified. We argue that there are no such reasons. In fact, we argue that promulgating the rule may actually decrease public trust in organ procurement procedures and medical institutions generally - even in states that do not permit capital punishment or voluntary active euthanasia. Finally, we examine our case against the dead donor rule in the light of common arguments for it. We find that these arguments are often misplaced - they do not support the dead donor rule. Instead, they support the quite different rule that patients should not be killed for their vital organs.

  14. Efficiency of box-traps and leg-hold traps with several bait types for capturing small carnivores (mammalia) in a disturbed area of southeastern Brazil.

    PubMed

    Michalski, Fernanda; Crawshaw, Peter G; Oliveira, Tadeu G de; Fabián, Marta E

    2007-03-01

    Capturing small carnivores is often necessary for obtaining key ecological data. We compared the efficiency of box and leg-hold traps, using live and dead bait, to capture six carnivore species (Herpailurus yagouaroundi (E. Geoffroyi, 1803), Leopardus tigrinus (Schreber, 1775), Nasua nasua (Linnaeus, 1766), Cerdocyon thous (Linnaeus, 1766), Eira barbara (Linnaeus, 1758), and Galictis cuja (Molina, 1782)). The use of leg-hold traps significantly increased the capture rate of carnivores (5.77%) and non-target species (non-carnivores, 11.54%). Dead bait significantly attracted more non-carnivores than carnivores and live bait was more efficient for capturing carnivores (2.56%) than non-carnivores (0.77%). Both box and leg-hold traps caused some minor injuries (swelling and claw loss). We provide recommendations for the ethical use of these trap and bait types.

  15. Hydro-Balanced Stuffing Box field test

    SciTech Connect

    Giangiacomo, L.A.

    1999-05-28

    The Hydro-Balanced Stuffing Box is a seal assembly for polished rod pumping installations commonly used in oil and gas pumping well installations to contain produced well fluids. The improved stuffing box was developed and patented by Harold H. Palmour of The Palmour Group of Livingston, TX. The stuffing box is designed to reduce the incidence of seal leakage and to utilize an environmentally safe fluid, so that if there is any leakage, environmental damage is reduced or eliminated. The unit was tested on two wells at the Rocky Mountain Oilfield Testing Center. During the test period, the performance of the stuffing box was measured by monitoring the pressure on the tubing and the inner chamber with a Barton Two-pen recorder. The amount of safe fluid consumed, fluid leakage at the top of the stuffing box, pressure supplied from the nitrogen bottle, ambient temperature, and polish rod temperature was recorded. The stuffing box is capable of providing a better seal between well fluids an d the environment than conventional stuffing boxes. It allows the polished rod to operate cooler and with lubrication, extending the life of the packing elements, and reducing the amount of attention required to prevent leakage.

  16. Genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase requires a sequence that is complementary to the binding site of the BMV helicase-like protein.

    PubMed

    Sivakumaran, K; Kao, C C

    2000-11-01

    Summary Initiation of genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) replicase in vitro requires a 26-nucleotide (nt) RNA sequence at the 3' end of the minus-strand RNA and a nontemplated nucleotide 3' of the initiation cytidylate [Sivakumaran, K. and Kao, C.C. (1999)J. Virol.64, 6415-6423]. At the 5' end of this RNA is a 9-nt sequence called the cB box, the complement of the previously defined B box. The cB box can not be functionally replaced by the B box and has specific positional and sequence requirements. The portion of the cB box that is required for RNA synthesis in vitro is well-conserved in species in the Bromoviridae family. An equivalent RNA from Cucumber mosaic virus was unable to direct efficient RNA synthesis by the BMV replicase until the cB box was positioned at the same site relative to the BMV RNA and guanylates were present at positions +6 and +7 from the initiation cytidylate. These results further define the elements required for the recognition and initiation of viral genomic plus-strand RNA synthesis and suggest that a sequence important for minus-strand RNA synthesis is also required for plus-strand RNA synthesis.

  17. Mutations in DDX59 Implicate RNA Helicase in the Pathogenesis of Orofaciodigital Syndrome

    PubMed Central

    Shamseldin, Hanan E.; Rajab, Anna; Alhashem, Amal; Shaheen, Ranad; Al-Shidi, Tarfa; Alamro, Rana; Al Harassi, Salma; Alkuraya, Fowzan S.

    2013-01-01

    Orofaciodigital syndrome (OFD) is a recognized clinical entity with core defining features in the mouth, face, and digits, in addition to various other features that have been proposed to define distinct subtypes. The three genes linked to OFD—OFD1, TMEM216, and TCTN3—play a role in ciliary biology, a finding consistent with the clinical overlap between OFD and other ciliopathies. Most autosomal-recessive cases of OFD, however, remain undefined genetically. In two multiplex consanguineous Arab families affected by OFD, we identified a tight linkage interval in chromosomal region 1q32.1. Exome sequencing revealed a different homozygous variant in DDX59 in each of the two families, and at least one of the two variants was accompanied by marked reduction in the level of DDX59. DDX59 encodes a relatively uncharacterized member of the DEAD-box-containing RNA helicase family of proteins, which are known to play a critical role in all aspects of RNA metabolism. We show that Ddx59 is highly enriched in its expression in the developing murine palate and limb buds. At the cellular level, we show that DDX59 is localized dynamically to the nucleus and the cytoplasm. Consistent with the absence of DDX59 representation in ciliome databases and our demonstration of its lack of ciliary localization, ciliogenesis appears to be intact in mutant fibroblasts but ciliary signaling appears to be impaired. Our data strongly implicate this RNA helicase family member in the pathogenesis of OFD, although the causal mechanism remains unclear. PMID:23972372

  18. HYDROGEN RETENTION IN METAL WASTE BOXES

    SciTech Connect

    MARUSICH, R.M.

    2004-11-18

    The Hanford Waste Management Project Master Documented Safety Analysis (MDSA) (HNF-14741,2003) identifies derived safety controls to prevent or mitigate the risks of a single- container deflagration during operations requiring moving, venting or opening transuranic (TRU)-waste containers. The issue is whether these safety controls are necessary for operations involving TRU-waste boxes that are being retrieved from burial at the Hanford Site. This paper investigates the potential for a deflagration hazard within these boxes and whether safety controls identified for drum deflagration hazards should be applied to operations involving these boxes.

  19. Eukaryotic tRNA paradox.

    PubMed

    Mitra, Sanga; Samadder, Arpa; Das, Pijush; Das, Smarajit; Chakrabarti, Jayprokas

    2015-01-01

    tRNAs are widely believed to segregate into two classes, I and II. Computational analysis of eukaryotic tRNA entries in Genomic tRNA Database, however, leads to new, albeit paradoxical, presence of more than a thousand class-I tRNAs with uncharacteristic long variable arms (V-arms), like in class-II. Out of 62,202 tRNAs from 69 eukaryotes, as many as 1431 class-I tRNAs have these novel extended V-arms, and we refer to them as paradoxical tRNAs (pxtRNAs). A great majority of these 1431 pxtRNA genes are located in intergenic regions, about 18% embedded in introns of genes or ESTs, and just one in 3'UTR. A check on the conservations of 2D and 3D base pairs for each position of these pxtRNAs reveals a few variations, but they seem to have almost all the known features (already known identity and conserved elements of tRNA). Analyses of the A-Box and B-Box of these pxtRNA genes in eukaryotes display salient deviations from the previously annotated conserved features of the standard promoters, whereas the transcription termination signals are just canonical and non-canonical runs of thymidine, similar to the ones in standard tRNA genes. There is just one such pxtRNA(ProAGG) gene in the entire human genome, and the availability of data allows epigenetic analysis of this human pxtRNA(ProAGG) in three different cell lines, H1 hESC, K562, and NHEK, to assess the level of its expression. Histone acetylation and methylation of this lone pxtRNA(ProAGG) gene in human differ from that of the nine standard human tRNA(ProAGG) genes. The V-arm nucleotide sequences and their secondary structures in pxtRNA differ from that of class-II tRNA. Considering these differences, hypotheses of alternative splicing, non-canonical intron and gene transfer are examined to partially improve the Cove scores of these pxtRNAs and to critically question their antecedence and novelty. PMID:25692737

  20. 36 CFR 1192.33 - Fare box.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 1192.33 Fare box. Where provided, the farebox shall be located as far forward as practicable...

  1. 36 CFR 1192.33 - Fare box.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 1192.33 Fare box. Where provided, the farebox shall be located as far forward as practicable...

  2. 36 CFR 1192.33 - Fare box.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 1192.33 Fare box. Where provided, the farebox shall be located as far forward as practicable...

  3. 36 CFR 1192.33 - Fare box.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION VEHICLES Buses, Vans and Systems § 1192.33 Fare box. Where provided, the farebox shall be located as far forward as practicable...

  4. Mystery Boxes, X Rays, and Radiology.

    ERIC Educational Resources Information Center

    Thomson, Norman

    2000-01-01

    Indicates the difficulties of teaching concepts beyond light and color and creating memorable learning experiences. Recommends sequential activities using the mystery box approach to explain how scientists and doctors use photon applications. (YDS)

  5. Aris-Taylor dispersion in tubes with dead ends.

    PubMed

    Dagdug, Leonardo; Berezhkovskii, Alexander M; Skvortsov, Alexei T

    2014-07-14

    This paper deals with transport of point Brownian particles in a cylindrical tube with dead ends in the presence of laminar flow of viscous fluid in the cylindrical part of the tube (Poiseuille flow). It is assumed that the dead ends are identical and are formed by spherical cavities connected to the cylindrical part of the tube by narrow necks. The focus is on the effective velocity and diffusivity of the particles as functions of the mean flow velocity and geometric parameter of the tube. Entering a dead end, the particle interrupts its propagation along the tube axis. Later it returns, and the axial motion continues. From the axial propagation point of view, the particle entry into a dead end and its successive return to the flow is equivalent to the particle reversible binding to the tube wall. The effect of reversible binding on the transport parameters has been previously studied assuming that the particle survival probability in the bound state decays as a single exponential. However, this is not the case when the particle enters a dead end, since escape from the dead end is a non-Markovian process. Our analysis of the problem consists of two steps: First, we derive expressions for the effective transport parameters in the general case of non-Markovian binding. Second, we find the effective velocity and diffusivity by substituting into these expressions known results for the moments of the particle lifetime in the dead end [L. Dagdug, A. M. Berezhkovskii, Yu. A. Makhnovskii, and V. Yu. Zitserman, J. Chem. Phys. 127, 224712 (2007)]. To check the accuracy of our theory, we compare its predictions with the values of the effective velocity and diffusivity obtained from Brownian dynamics simulations. The comparison shows excellent agreement between the theoretical predictions and numerical results. PMID:25028036

  6. RNA epigenetics

    PubMed Central

    Liu, Nian; Pan, Tao

    2014-01-01

    Summary Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modification. PMID:24768686

  7. BOX DIMENSIONS OF α-FRACTAL FUNCTIONS

    NASA Astrophysics Data System (ADS)

    Akhtar, Md. Nasim; Prasad, M. Guru Prem; Navascués, M. A.

    2016-08-01

    The box dimension of the graph of non-affine, continuous, nowhere differentiable function fα which is a fractal analogue of a continuous function f corresponding to a certain iterated function system (IFS), is investigated in the present paper. The estimates for box dimension of the graph of α-fractal function fα for equally spaced as well as arbitrary data sets are found.

  8. BOX-DEATH HOLLOW ROADLESS AREA, UTAH.

    USGS Publications Warehouse

    Weir, Gordon W.; Lane, Michael

    1984-01-01

    Geologic mapping, geochemical sampling, and a search for prospects and mineralized rock in the Box-Death Hollow Roadless Area, Utah indicate that there is little promise for the occurrence of mineral or energy resources in the area. Additional exploratory drilling by industry seems warranted if wells elsewhere in the region find oil or gas in strata as yet untested in the Box-Death Hollow Roadless Area.

  9. Glove box on vehicular instrument panel

    DOEpatents

    Atarashi, Kazuya

    1985-01-01

    A glove box for the upper surface of an automobile dashboard whereby it may be positioned close to the driver. The glove box lid is pivotally supported by arms extending down either side to swing forwardly for opening. A hook is pivotally support adjacent an arm and weighted to swing into engagement with the arm to prevent opening of the lid during abrupt deceleration. A toggle spring assists in maintaining the lid in either the open or closed position.

  10. Fibrillarin and Nop56 interact before being co-assembled in box C/D snoRNPs

    SciTech Connect

    Lechertier, Tanguy; Grob, Alice; Hernandez-Verdun, Daniele

    2009-04-01

    Small nucleolar RNAs play crucial roles in ribosome biogenesis. They guide folding, site-specific nucleotide modifications and participate in cleavage of precursor ribosomal RNAs. To better understand how the biogenesis of the box C/D small nucleolar RNPs (snoRNPs) occur in a cellular context, we used a new approach based on the possibility of relocalizing a given nuclear complex by adding an affinity tag for B23 to one component of this complex. We selectively delocalized each core box C/D protein, namely 15.5kD, Nop56, Nop58 and fibrillarin, and analyzed the effect of such changes on other components of the box C/D snoRNPs. We show that modifying the localization and the mobility of core box C/D proteins impairs their association with box C/D snoRNPs. In addition, we demonstrate that fibrillarin and Nop56 directly interact in vivo. This interaction, indispensable for the association of both proteins with the box C/D snoRNPs, does not involve the glycine- and arginine-rich domain or the RNA-binding domain but the alpha-helix domain of fibrillarin. In addition, no RNA seems required to maintain fibrillarin-Nop56 interaction.

  11. Amateur boxing: physical and physiological attributes.

    PubMed

    Chaabène, Helmi; Tabben, Montassar; Mkaouer, Bessem; Franchini, Emerson; Negra, Yassine; Hammami, Mehrez; Amara, Samiha; Chaabène, Raja Bouguezzi; Hachana, Younés

    2015-03-01

    Boxing is one of the oldest combat sports. The aim of the current review is to critically analyze the amateur boxer's physical and physiological characteristics and to provide practical recommendations for training as well as new areas of scientific research. High-level male and female boxers show a propensity for low body fat levels. Although studies on boxer somatotypes are limited, the available information shows that elite-level male boxers are characterized by a higher proportion of mesomorphy with a well-developed muscle mass and a low body fat level. To help support the overall metabolic demands of a boxing match and to accelerate the recovery process between rounds, athletes of both sexes require a high level of cardiorespiratory fitness. International boxers show a high peak and mean anaerobic power output. Muscle strength in both the upper and lower limbs is paramount for a fighter's victory and is one of the keys to success in boxing. As boxing punches are brief actions and very dynamic, high-level boxing performance requires well-developed muscle power in both the upper and lower limbs. Albeit limited, the available studies reveal that isometric strength is linked to high-level boxing performance. Future investigations into the physical and physiological attributes of boxers are required to enrich the current data set and to help create a suitable training program. PMID:25358529

  12. HYDROGEN AND VOC RETENTION IN WASTE BOXES

    SciTech Connect

    PACE ME; MARUSICH RM

    2008-11-21

    The Hanford Waste Management Project Master Documented Safety Analysis (MDSA) (HNF-14741, 2003) identifies derived safety controls to prevent or mitigate the risks of a single-container deflagration during operations requiring moving, venting or opening transuranic (TRU)-waste containers. The issue is whether these safety controls are necessary for operations involving TRU-waste boxes that are being retrieved from burial at the Hanford Site. This paper investigates the potential for a deflagration hazard within these boxes and whether safety controls identified for drum deflagration hazards should be applied to operations involving these boxes. The study evaluates the accumulation of hydrogen and VOCs within the waste box and the transport of these gases and vapors out of the waste box. To perform the analysis, there were numerous and major assumptions made regarding the generation rate and the transport pathway dimensions and their number. Since there is little actual data with regards to these assumptions, analyses of three potential configurations were performed to obtain some indication of the bounds of the issue (the concentration of hydrogen or flammable VOCs within a waste box). A brief description of each of the three cases along with the results of the analysis is summarized.

  13. Exon ligation is proofread by the DExD/H-box ATPase Prp22p.

    PubMed

    Mayas, Rabiah M; Maita, Hiroshi; Staley, Jonathan P

    2006-06-01

    To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3' splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.

  14. Teleseismic travel times residuals across the Dead Sea basin

    NASA Astrophysics Data System (ADS)

    Hofstetter, Rami; Dorbath, Catherine

    2014-05-01

    New findings of the structure of the Dead Sea sedimentary basin and its eastern and western bordering regions were obtained by applying P and PKP wave relative travel time residuals of 644 teleseisms, as recorded by the DESIRE portable seismic network in the Dead Sea basin and its outskirts. The Lisan is characterized by relatively small teleseismic travel time residuals of about 0.14 sec, in the latitude range of 31.220-31.370 and longitude of 35.500, slowly degrading towards west. The largest teleseismic travel time residuals are in the southern Dead Sea basin, south of the Lisan in the latitude range of 31.050-31.150 and along longitude 35.450 and continuing southward towards Amatzyahu Fault, reaching values of 0.3 to 0.4 sec. We get small positive residual in the Amatzyahu Fault and small negative residual south of it marking probably the southern end of the Dead Sea basin. East and west of the Dead Sea basin the mean teleseismic travel time residuals are negative having an over whole average of -0.35 sec and -0.45 sec, respectively. Using the teleseismic residuals we estimate the horizontal dimensions of the Lisan salt diapir to be 20 km X 12 km at its widest place and a maximal thickness of about 7.2 km. The thickness of the Mt. Sodom salt diapir is estimated as 6.2 km.

  15. The Dead Sea, The Lake and Its Setting

    NASA Astrophysics Data System (ADS)

    Brink, Uri ten

    I cannot think of a subject more befitting the description of interdisciplinary research with societal relevance than the study of the Dead Sea, a terminal lake of the Jordan River in Israel and Jordan. The scientific study of the Dead Sea is intimately connected with politics, religion, archeology, economic development, tourism, and environmental change.The Dead Sea is a relatively closed geologic and limnologic system with drastic physical changes often occurring on human timescales and with a long human history to observe these changes. Research in this unique area covers diverse aspects such as active subsidence and deformation along strike-slip faults; vertical stratification and stability of the water column; physical properties of extremely saline and dense (1234 kg/m3) water; spontaneous precipitation of minerals in an oversaturated environment; origin of the unusual chemical composition of the brine; existence of life in extreme environments; use of lake level fluctuations as a paleoclimatic indicator; and effects on the environment of human intervention versus natural climatic variability. Although the Dead Sea covers a small area on a global scale, it is nevertheless one of the largest natural laboratories for these types of research on Earth. These reasons make the Dead Sea a fascinating topic for the curious mind.

  16. Box C/D guide RNAs recognize a maximum of 10 nt of substrates.

    PubMed

    Yang, Zuxiao; Lin, Jinzhong; Ye, Keqiong

    2016-09-27

    Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D' and boxes C' to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide-substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C' side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.

  17. Zombie Vortex Instability. I. A Purely Hydrodynamic Instability to Resurrect the Dead Zones of Protoplanetary Disks

    NASA Astrophysics Data System (ADS)

    Marcus, Philip S.; Pei, Suyang; Jiang, Chung-Hsiang; Barranco, Joseph A.; Hassanzadeh, Pedram; Lecoanet, Daniel

    2015-07-01

    There is considerable interest in hydrodynamic instabilities in dead zones of protoplanetary disks as a mechanism for driving angular momentum transport and as a source of particle-trapping vortices to mix chondrules and incubate planetesimal formation. We present simulations with a pseudo-spectral anelastic code and with the compressible code Athena, showing that stably stratified flows in a shearing, rotating box are violently unstable and produce space-filling, sustained turbulence dominated by large vortices with Rossby numbers of order ˜0.2–0.3. This Zombie Vortex Instability (ZVI) is observed in both codes and is triggered by Kolmogorov turbulence with Mach numbers less than ˜0.01. It is a common view that if a given constant density flow is stable, then stable vertical stratification should make the flow even more stable. Yet, we show that sufficient vertical stratification can be unstable to ZVI. ZVI is robust and requires no special tuning of boundary conditions, or initial radial entropy or vortensity gradients (though we have studied ZVI only in the limit of infinite cooling time). The resolution of this paradox is that stable stratification allows for a new avenue to instability: baroclinic critical layers. ZVI has not been seen in previous studies of flows in rotating, shearing boxes because those calculations frequently lacked vertical density stratification and/or sufficient numerical resolution. Although we do not expect appreciable angular momentum transport from ZVI in the small domains in this study, we hypothesize that ZVI in larger domains with compressible equations may lead to angular transport via spiral density waves.

  18. Zombie Vortex Instability. I. A Purely Hydrodynamic Instability to Resurrect the Dead Zones of Protoplanetary Disks

    NASA Astrophysics Data System (ADS)

    Marcus, Philip S.; Pei, Suyang; Jiang, Chung-Hsiang; Barranco, Joseph A.; Hassanzadeh, Pedram; Lecoanet, Daniel

    2015-07-01

    There is considerable interest in hydrodynamic instabilities in dead zones of protoplanetary disks as a mechanism for driving angular momentum transport and as a source of particle-trapping vortices to mix chondrules and incubate planetesimal formation. We present simulations with a pseudo-spectral anelastic code and with the compressible code Athena, showing that stably stratified flows in a shearing, rotating box are violently unstable and produce space-filling, sustained turbulence dominated by large vortices with Rossby numbers of order ˜0.2-0.3. This Zombie Vortex Instability (ZVI) is observed in both codes and is triggered by Kolmogorov turbulence with Mach numbers less than ˜0.01. It is a common view that if a given constant density flow is stable, then stable vertical stratification should make the flow even more stable. Yet, we show that sufficient vertical stratification can be unstable to ZVI. ZVI is robust and requires no special tuning of boundary conditions, or initial radial entropy or vortensity gradients (though we have studied ZVI only in the limit of infinite cooling time). The resolution of this paradox is that stable stratification allows for a new avenue to instability: baroclinic critical layers. ZVI has not been seen in previous studies of flows in rotating, shearing boxes because those calculations frequently lacked vertical density stratification and/or sufficient numerical resolution. Although we do not expect appreciable angular momentum transport from ZVI in the small domains in this study, we hypothesize that ZVI in larger domains with compressible equations may lead to angular transport via spiral density waves.

  19. On the dynamics of planetesimals embedded in turbulent protoplanetary discs with dead zones

    NASA Astrophysics Data System (ADS)

    Gressel, Oliver; Nelson, Richard P.; Turner, Neal J.

    2011-08-01

    Accretion in protoplanetary discs is thought to be driven by magnetohydrodynamic (MHD) turbulence via the magnetorotational instability. Recent work has shown that a planetesimal swarm embedded in a fully turbulent disc is subject to strong excitation of the velocity dispersion, leading to collisional destruction of bodies with radii Rp < 100 km. Significant diffusion of planetesimal semimajor axes also arises, leading to large-scale spreading of the planetesimal population throughout the inner regions of the protoplanetary disc, in apparent contradiction of constraints provided by the distribution of asteroids within the asteroid belt. In this paper, we examine the dynamics of planetesimals embedded in vertically stratified turbulent discs, with and without dead zones. Our main aims are to examine the turbulent excitation of the velocity dispersion, and the radial diffusion, of planetesimals in these discs. We employ 3D MHD simulations using the shearing box approximation, along with an equilibrium chemistry model that is used to calculate the ionization fraction of the disc gas as a function of time and position. Ionization is assumed to arise because of stellar X-rays, galactic cosmic rays and radioactive nuclei. In agreement with our previous study, we find that planetesimals in fully turbulent discs develop large random velocities that will lead to collisional destruction/erosion for bodies with sizes below 100 km, and undergo radial diffusion on a scale ˜2.5 au over a 5 Myr disc lifetime. But planetesimals in a dead zone experience a much reduced excitation of their random velocities, and equilibrium velocity dispersions lie between the disruption thresholds for weak and strong aggregates for sizes Rp≤ 100 km. We also find that radial diffusion occurs over a much reduced length-scale ˜0.25 au over the disc lifetime, this being consistent with Solar system constraints. We conclude that planetesimal growth via mutual collisions between smaller bodies cannot

  20. The Dead Sea Transform and the Dead Sea Basin - Structure and dynamics

    NASA Astrophysics Data System (ADS)

    Weber, M.; Desire Groups, D A

    2004-12-01

    DESERT and DESIRE, two multi-national, interdisciplinary research efforts by teams from Germany, Israel, Jordan and Palestine focused on the Dead Sea Transform (DST) and the Dead Sea Basin (DSB), respectively. The DST has accommodated left-lateral transform motion of 105 km between the African and Arabian plates since early Miocene (ca. 20 My), creating during this process also the prime example of a pull-apart basin, the DSB. Within DESERT the DST segment between the Dead Sea and the Red Sea called Arava/Araba Fault (AF) was studied with the following results. On plate tectonic scale the AF is a narrow, sub-vertical zone cutting through crust and lithosphere to more than 50 km depth, while the Moho depth increases smoothly from 26 km to 39 km from W to E under the DST. Several faults exist in the upper crust in a ca. 40 km wide zone around the AF, but none has kilometer-size zones of decreased seismic velocities/zones of high electrical conductivities typical for damage zones. Across the sub-vertical AF abrupt changes in lithology can be identified to a depth of 4 kilometers. The AF also acts as a barrier to fluids. The AF is the main active fault of the DST system but it has only accommodated a limited part (up to 60 km) of the overall 105 km of sinistral plate motion. Now inactive fault strands in the vicinity of the present day AF took up lateral motion until about 5 Ma ago, when the main, active fault trace shifted ca. 1 km westward to its present position. In the top few hundred meters of the AF a locally transpressional regime occurs in a 100 to 300 m wide zone of deformed and displaced material, bordered by sub-parallel faults forming positive flower structures. The damage zones of the individual faults are only 5 to 20 m wide. This narrow width is significantly smaller than at other major strike-slip faults of similar magnitude. Most of these findings are corroborated by thermo-mechanical modeling that show shear deformation in the lithosphere under the

  1. The Dead Sea Transform and the Dead Sea Basin - Structure and dynamics

    NASA Astrophysics Data System (ADS)

    Weber, M.; Desire Groups, D A

    2007-12-01

    DESERT and DESIRE, two multi-national, interdisciplinary research efforts by teams from Germany, Israel, Jordan and Palestine focused on the Dead Sea Transform (DST) and the Dead Sea Basin (DSB), respectively. The DST has accommodated left-lateral transform motion of 105 km between the African and Arabian plates since early Miocene (ca. 20 My), creating during this process also the prime example of a pull-apart basin, the DSB. Within DESERT the DST segment between the Dead Sea and the Red Sea called Arava/Araba Fault (AF) was studied with the following results. On plate tectonic scale the AF is a narrow, sub-vertical zone cutting through crust and lithosphere to more than 50 km depth, while the Moho depth increases smoothly from 26 km to 39 km from W to E under the DST. Several faults exist in the upper crust in a ca. 40 km wide zone around the AF, but none has kilometer-size zones of decreased seismic velocities/zones of high electrical conductivities typical for damage zones. Across the sub-vertical AF abrupt changes in lithology can be identified to a depth of 4 kilometers. The AF also acts as a barrier to fluids. The AF is the main active fault of the DST system but it has only accommodated a limited part (up to 60 km) of the overall 105 km of sinistral plate motion. Now inactive fault strands in the vicinity of the present day AF took up lateral motion until about 5 Ma ago, when the main, active fault trace shifted ca. 1 km westward to its present position. In the top few hundred meters of the AF a locally transpressional regime occurs in a 100 to 300 m wide zone of deformed and displaced material, bordered by sub-parallel faults forming positive flower structures. The damage zones of the individual faults are only 5 to 20 m wide. This narrow width is significantly smaller than at other major strike-slip faults of similar magnitude. Most of these findings are corroborated by thermo-mechanical modeling that show shear deformation in the lithosphere under the

  2. 46 CFR 78.47-10 - Manual alarm boxes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 3 2011-10-01 2011-10-01 false Manual alarm boxes. 78.47-10 Section 78.47-10 Shipping... and Emergency Equipment, Etc. § 78.47-10 Manual alarm boxes. (a) In all new installations, manual alarm boxes shall be clearly and permanently marked “IN CASE OF FIRE BREAK GLASS.” Existing boxes not...

  3. 48 CFR 908.7118 - Rental of post office boxes.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... boxes. 908.7118 Section 908.7118 Federal Acquisition Regulations System DEPARTMENT OF ENERGY COMPETITION... post office boxes. DOE offices and authorized contractors may rent post office boxes on an annual basis... the whole period or at the beginning of each quarter in which the box is to be used....

  4. 48 CFR 908.7118 - Rental of post office boxes.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... boxes. 908.7118 Section 908.7118 Federal Acquisition Regulations System DEPARTMENT OF ENERGY COMPETITION... post office boxes. DOE offices and authorized contractors may rent post office boxes on an annual basis... the whole period or at the beginning of each quarter in which the box is to be used....

  5. 49 CFR 178.514 - Standards for plywood boxes.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false Standards for plywood boxes. 178.514 Section 178...-bulk Performance-Oriented Packaging Standards § 178.514 Standards for plywood boxes. (a) The identification code for a plywood box is 4D. (b) Construction requirements for plywood boxes are as follows:...

  6. 46 CFR 78.47-10 - Manual alarm boxes.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 3 2013-10-01 2013-10-01 false Manual alarm boxes. 78.47-10 Section 78.47-10 Shipping... and Emergency Equipment, Etc. § 78.47-10 Manual alarm boxes. (a) In all new installations, manual alarm boxes shall be clearly and permanently marked “IN CASE OF FIRE BREAK GLASS.” Existing boxes not...

  7. 49 CFR 178.514 - Standards for plywood boxes.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false Standards for plywood boxes. 178.514 Section 178...-bulk Performance-Oriented Packaging Standards § 178.514 Standards for plywood boxes. (a) The identification code for a plywood box is 4D. (b) Construction requirements for plywood boxes are as follows:...

  8. 48 CFR 908.7118 - Rental of post office boxes.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... boxes. 908.7118 Section 908.7118 Federal Acquisition Regulations System DEPARTMENT OF ENERGY COMPETITION... post office boxes. DOE offices and authorized contractors may rent post office boxes on an annual basis... the whole period or at the beginning of each quarter in which the box is to be used....

  9. 46 CFR 78.47-10 - Manual alarm boxes.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 3 2012-10-01 2012-10-01 false Manual alarm boxes. 78.47-10 Section 78.47-10 Shipping... and Emergency Equipment, Etc. § 78.47-10 Manual alarm boxes. (a) In all new installations, manual alarm boxes shall be clearly and permanently marked “IN CASE OF FIRE BREAK GLASS.” Existing boxes not...

  10. 46 CFR 78.47-10 - Manual alarm boxes.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 3 2014-10-01 2014-10-01 false Manual alarm boxes. 78.47-10 Section 78.47-10 Shipping... and Emergency Equipment, Etc. § 78.47-10 Manual alarm boxes. (a) In all new installations, manual alarm boxes shall be clearly and permanently marked “IN CASE OF FIRE BREAK GLASS.” Existing boxes not...

  11. 48 CFR 908.7118 - Rental of post office boxes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... boxes. 908.7118 Section 908.7118 Federal Acquisition Regulations System DEPARTMENT OF ENERGY COMPETITION... post office boxes. DOE offices and authorized contractors may rent post office boxes on an annual basis... the whole period or at the beginning of each quarter in which the box is to be used....

  12. 48 CFR 908.7118 - Rental of post office boxes.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... boxes. 908.7118 Section 908.7118 Federal Acquisition Regulations System DEPARTMENT OF ENERGY COMPETITION... post office boxes. DOE offices and authorized contractors may rent post office boxes on an annual basis... the whole period or at the beginning of each quarter in which the box is to be used....

  13. 49 CFR 178.514 - Standards for plywood boxes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false Standards for plywood boxes. 178.514 Section 178...-bulk Performance-Oriented Packaging Standards § 178.514 Standards for plywood boxes. (a) The identification code for a plywood box is 4D. (b) Construction requirements for plywood boxes are as follows:...

  14. 49 CFR 230.101 - Steam locomotive driving journal boxes.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Steam locomotive driving journal boxes. 230.101... Locomotives and Tenders Running Gear § 230.101 Steam locomotive driving journal boxes. (a) Driving journal boxes. Driving journal boxes shall be maintained in a safe and suitable condition for service. Not...

  15. 49 CFR 230.101 - Steam locomotive driving journal boxes.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 4 2013-10-01 2013-10-01 false Steam locomotive driving journal boxes. 230.101... Locomotives and Tenders Running Gear § 230.101 Steam locomotive driving journal boxes. (a) Driving journal boxes. Driving journal boxes shall be maintained in a safe and suitable condition for service. Not...

  16. 49 CFR 230.101 - Steam locomotive driving journal boxes.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Steam locomotive driving journal boxes. 230.101... Locomotives and Tenders Running Gear § 230.101 Steam locomotive driving journal boxes. (a) Driving journal boxes. Driving journal boxes shall be maintained in a safe and suitable condition for service. Not...

  17. 49 CFR 230.101 - Steam locomotive driving journal boxes.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Steam locomotive driving journal boxes. 230.101... Locomotives and Tenders Running Gear § 230.101 Steam locomotive driving journal boxes. (a) Driving journal boxes. Driving journal boxes shall be maintained in a safe and suitable condition for service. Not...

  18. 49 CFR 230.101 - Steam locomotive driving journal boxes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Steam locomotive driving journal boxes. 230.101... Locomotives and Tenders Running Gear § 230.101 Steam locomotive driving journal boxes. (a) Driving journal boxes. Driving journal boxes shall be maintained in a safe and suitable condition for service. Not...

  19. RNA-guided isomerization of uridine to pseudouridine—pseudouridylation

    PubMed Central

    Yu, Yi-Tao; Meier, U Thomas

    2014-01-01

    Box H/ACA ribonucleoproteins (RNPs), each consisting of one unique guide RNA and 4 common core proteins, constitute a family of complex enzymes that catalyze, in an RNA-guided manner, the isomerization of uridines to pseudouridines (Ψs) in RNAs, a reaction known as pseudouridylation. Over the years, box H/ACA RNPs have been extensively studied revealing many important aspects of these RNA modifying machines. In this review, we focus on the composition, structure, and biogenesis of H/ACA RNPs. We explain the mechanism of how this enzyme family recognizes and specifies its target uridine in a substrate RNA. We discuss the substrates of box H/ACA RNPs, focusing on rRNA (rRNA) and spliceosomal small nuclear RNA (snRNA). We describe the modification product Ψ and its contribution to RNA function. Finally, we consider possible mechanisms of the bone marrow failure syndrome dyskeratosis congenita and of prostate and other cancers linked to mutations in H/ACA RNPs. PMID:25590339

  20. The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

    PubMed Central

    Padilla-Mejía, Norma E.; Florencio-Martínez, Luis E.; Moreno-Campos, Rodrigo; Vizuet-de-Rueda, Juan C.; Cevallos, Ana M.; Hernández-Rivas, Rosaura; Manning-Cela, Rebeca

    2014-01-01

    Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes. PMID:25548151