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Sample records for elongation factor nelf

  1. Cellular dynamics of the negative transcription elongation factor NELF

    SciTech Connect

    Yung, Tetsu M.C.; Narita, Takashi; Komori, Toshiharu; Yamaguchi, Yuki; Handa, Hiroshi

    2009-06-10

    Negative Elongation Factor (NELF) is a transcription factor discovered based on its biochemical activity to suppress transcription elongation, and has since been implicated in various diseases ranging from neurological disorders to cancer. Besides its role in promoter-proximal pausing of RNA polymerase II during early stages of transcription, recently we found that it also plays important roles in the 3'-end processing of histone mRNA. Furthermore, NELF has been found to form a distinct subnuclear structure, which we named NELF bodies. These recent developments point to a wide range of potential functions for NELF, and, as most studies on NELF thus far had been carried out in vitro, here, we prepared a complete set of fusion protein constructs of NELF subunits and carried out a general cell biological study of the intracellular dynamics of NELF. Our data show that NELF subunits exhibit highly specific subcellular localizations, such as in NELF bodies or in midbodies, and some shuttle actively between the nucleus and cytoplasm. We further show that loss of NELF from cells can lead to enlarged and/or multiple nuclei. This work serves as a foundation and starting point for further cell biological investigations of NELF in the future.

  2. Negative elongation factor NELF controls transcription of immediate early genes in a stimulus-specific manner

    SciTech Connect

    Fujita, Toshitsugu; Piuz, Isabelle; Schlegel, Werner

    2009-01-15

    The transcription rate of immediate early genes (IEGs) is controlled directly by transcription elongation factors at the transcription elongation step. Negative elongation factor (NELF) and 5,6-dichloro-1-{beta}-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) stall RNA polymerase II (pol II) soon after transcription initiation. Upon induction of IEG transcription, DSIF is converted into an accelerator for pol II elongation. To address whether and how NELF as well as DSIF controls overall IEG transcription, its expression was reduced using stable RNA interference in GH4C1 cells. NELF knock-down reduced thyrotropin-releasing hormone (TRH)-induced transcription of the IEGs c-fos, MKP-1, and junB. In contrast, epidermal growth factor (EGF)-induced transcription of these IEGs was unaltered or even slightly increased by NELF knock-down. Thus, stable knock-down of NELF affects IEG transcription stimulation-specifically. Conversely, DSIF knock-down reduced both TRH- and EGF-induced transcription of the three IEGs. Interestingly, TRH-induced activation of the MAP kinase pathway, a pathway essential for transcription of the three IEGs, was down-regulated by NELF knock-down. Thus, stable knock-down of NELF, by modulating intracellular signaling pathways, caused stimulation-specific loss of IEG transcription. These observations indicate that NELF controls overall IEG transcription via multiple mechanisms both directly and indirectly.

  3. NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila▿ †

    PubMed Central

    Lee, Chanhyo; Li, Xiaoyong; Hechmer, Aaron; Eisen, Michael; Biggin, Mark D.; Venters, Bryan J.; Jiang, Cizhong; Li, Jian; Pugh, B. Franklin; Gilmour, David S.

    2008-01-01

    Recent analyses of RNA polymerase II (Pol II) revealed that Pol II is concentrated at the promoters of many active and inactive genes. NELF causes Pol II to pause in the promoter-proximal region of the hsp70 gene in Drosophila melanogaster. In this study, genome-wide location analysis (chromatin immunoprecipitation-microarray chip [ChIP-chip] analysis) revealed that NELF is concentrated at the 5′ ends of 2,111 genes in Drosophila cells. Permanganate genomic footprinting was used to determine if paused Pol II colocalized with NELF. Forty-six of 56 genes with NELF were found to have paused Pol II. Pol II pauses 30 to 50 nucleotides downstream from transcription start sites. Analysis of DNA sequences in the vicinity of paused Pol II identified a conserved DNA sequence that probably associates with TFIID but detected no evidence of RNA secondary structures or other conserved sequences that might directly control elongation. ChIP-chip experiments indicate that GAGA factor associates with 39% of the genes that have NELF. Surprisingly, NELF associates with almost one-half of the most highly expressed genes, indicating that NELF is not necessarily a repressor of gene expression. NELF-associated pausing of Pol II might be an obligatory but sometimes transient checkpoint during the transcription cycle. PMID:18332113

  4. Architecture and RNA binding of the human negative elongation factor

    PubMed Central

    Vos, Seychelle M; Pöllmann, David; Caizzi, Livia; Hofmann, Katharina B; Rombaut, Pascaline; Zimniak, Tomasz; Herzog, Franz; Cramer, Patrick

    2016-01-01

    Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF. DOI: http://dx.doi.org/10.7554/eLife.14981.001 PMID:27282391

  5. Negative elongation factor controls energy homeostasis in cardiomyocytes.

    PubMed

    Pan, Haihui; Qin, Kunhua; Guo, Zhanyong; Ma, Yonggang; April, Craig; Gao, Xiaoli; Andrews, Thomas G; Bokov, Alex; Zhang, Jianhua; Chen, Yidong; Weintraub, Susan T; Fan, Jian-Bing; Wang, Degeng; Hu, Yanfen; Aune, Gregory J; Lindsey, Merry L; Li, Rong

    2014-04-10

    Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes. PMID:24656816

  6. A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

    PubMed Central

    Palangat, Murali; Renner, Dan B.; Price, David H.; Landick, Robert

    2005-01-01

    Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-β-d-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires ≥18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of back-tracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription. PMID:16214896

  7. NF-κB-repressing factor phosphorylation regulates transcription elongation via its interactions with 5'→3' exoribonuclease 2 and negative elongation factor.

    PubMed

    Rother, Sascha; Bartels, Myriam; Schweda, Aike Torben; Resch, Klaus; Pallua, Norbert; Nourbakhsh, Mahtab

    2016-01-01

    NF-κB-repressing factor (NKRF) inhibits transcription elongation by binding to specific sequences in target promoters. Stimuli such as IL-1 have been shown to overcome this inhibitory action and enable the resumption of transcription elongation machinery by an unknown mechanism. Using mass spectrometry and in vitro phosphorylation analyses, we demonstrate that NKRF is phosphorylated within 3 different domains in unstimulated HeLa cells. Phosphoamino acid mapping and mutation analysis of NKRF further suggest that only Ser phosphorylation within aa 421-429 is regulated by IL-1 stimulation. In copurification studies, aa 421-429 is required for interactions between NKRF, 5'→3' exoribonuclease 2 (XRN2) and the negative elongation factor (NELF)-E in HeLa cells. Chromatin immunoprecipitation experiments further show that IL-1 stimulation leads to decrease in NKRF aa 421-429 phosphorylation and dissociation of NELF-E and XRN2 by concomitant resumption of transcription elongation of a synthetic reporter or the endogenous NKRF target gene, IL-8. Together, NKRF phosphorylation modulates promoter-proximal transcription elongation of NF-κB/NKRF-regulated genes via direct interactions with elongation complex in response to specific stimuli.

  8. Negative Elongation Factor Is Required for the Maintenance of Proviral Latency but Does Not Induce Promoter-Proximal Pausing of RNA Polymerase II on the HIV Long Terminal Repeat

    PubMed Central

    Jadlowsky, Julie K.; Wong, Julian Y.; Graham, Amy C.; Dobrowolski, Curtis; Devor, Renee L.; Adams, Mark D.; Fujinaga, Koh

    2014-01-01

    The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5′ long terminal repeat (LTR) but not on the 3′ LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses. PMID:24636995

  9. NELF knockout is associated with impaired pubertal development and subfertility.

    PubMed

    Quaynor, Samuel D; Ko, Eun Kyung; Chorich, Lynn P; Sullivan, Megan E; Demir, Durkadin; Waller, Jennifer L; Kim, Hyung-Goo; Cameron, Richard S; Layman, Lawrence C

    2015-05-15

    Puberty and reproduction require proper signaling of the hypothalamic-pituitary-gonadal axis controlled by gonadotropin-releasing hormone (GnRH) neurons, which arise in the olfactory placode region and migrate along olfactory axons to the hypothalamus. Factors adversely affecting GnRH neuron specification, migration, and function lead to delayed puberty and infertility. Nasal embryonic luteinizing hormone-releasing factor (NELF) is a predominantly nuclear protein. NELF mutations have been demonstrated in patients with hypogonadotropic hypogonadism, but biallelic mutations are rare and heterozygous NELF mutations typically co-exist with mutations in another gene. Our previous studies in immortalized GnRH neurons supported a role for NELF in GnRH neuron migration. To better understand the physiology of NELF, a homozygous Nelf knockout (KO) mouse model was generated. Our findings indicate that female Nelf KO mice have delayed vaginal opening but no delay in time to first estrus, decreased uterine weight, and reduced GnRH neuron number. In contrast, male mice were normal at puberty. Both sexes of mice had impaired fertility manifested as reduced mean litter size. These data support that NELF has important reproductive functions. The milder than expected phenotype of KO mice also recapitulates the human phenotype since heterozygous NELF mutations usually require an additional mutation in a second gene to result in hypogonadotropic hypogonadism.

  10. CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription.

    PubMed

    Laitem, Clélia; Zaborowska, Justyna; Tellier, Michael; Yamaguchi, Yuki; Cao, Qingfu; Egloff, Sylvain; Handa, Hiroshi; Murphy, Shona

    2015-01-01

    CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.

  11. NELF is a nuclear protein involved in hypothalamic GnRH neuronal migration.

    PubMed

    Xu, Ning; Bhagavath, Balasubramanian; Kim, Hyung-Goo; Halvorson, Lisa; Podolsky, Robert S; Chorich, Lynn P; Prasad, Puttur; Xiong, Wen-Cheng; Cameron, Richard S; Layman, Lawrence C

    2010-05-01

    Nasal embryonic LHRH factor (NELF) has been hypothesized to participate in the migration of GnRH and olfactory neurons into the forebrain, a prerequisite for normal hypothalamic-pituitary-gonadal function in puberty and reproduction. However, the biological functions of NELF, which has no homology to any human protein, remain largely elusive. Although mRNA expression did not differ, NELF protein expression was greater in migratory than postmigratory GnRH neurons. Pituitary Nelf mRNA expression was also observed and increased 3-fold after exogenous GnRH administration. Contrary to a previous report, NELF displayed predominant nuclear localization in GnRH neurons, confirmed by mutagenesis of a putative nuclear localization signal resulting in impaired nuclear expression. NELF knockdown impaired GnRH neuronal migration of NLT cells in vitro. These findings and the identification of two putative zinc fingers suggest that NELF could be a transcription factor. Collectively, our findings implicate NELF as a nuclear protein involved in the developmental function of the reproductive axis.

  12. Transcription factor Sp3 represses expression of p21CIP¹ via inhibition of productive elongation by RNA polymerase II.

    PubMed

    Valin, Alvaro; Ouyang, Jian; Gill, Grace

    2013-04-01

    Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle inhibitor is regulated at the level of transcription elongation. While many transcriptional activators have been shown to stimulate elongation, the mechanisms by which promoter-specific repressors regulate pausing and elongation by RNA polymerase II (RNA PolII) are not well described. Here we report that the transcription factor Sp3 inhibits basal p21(CIP1) gene expression by promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA levels and reduced occupancy of the negative elongation factor (NELF) at the p21(CIP1) promoter, although the level of binding of the positive transcription elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated with increased H3K36me3 and H2Bub1, two histone modifications associated with transcription elongation. Further, Sp3 was shown to promote the binding of protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10 phosphorylation, a finding consistent with Sp3-dependent regulation of the local balance between kinase and phosphatase activities. Analysis of other targets of Sp3-mediated repression suggests that, in addition to previously described SUMO modification-dependent chromatin-silencing mechanisms, inhibition of the transition of paused RNA PolII to productive elongation, described here for p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes gene expression.

  13. Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation.

    PubMed

    Gibson, Bryan A; Zhang, Yajie; Jiang, Hong; Hussey, Kristine M; Shrimp, Jonathan H; Lin, Hening; Schwede, Frank; Yu, Yonghao; Kraus, W Lee

    2016-07-01

    Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

  14. Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation.

    PubMed

    Gibson, Bryan A; Zhang, Yajie; Jiang, Hong; Hussey, Kristine M; Shrimp, Jonathan H; Lin, Hening; Schwede, Frank; Yu, Yonghao; Kraus, W Lee

    2016-07-01

    Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals. PMID:27256882

  15. Actin nucleation and elongation factors: mechanisms and interplay.

    PubMed

    Chesarone, Melissa A; Goode, Bruce L

    2009-02-01

    Cells require actin nucleators to catalyze the de novo assembly of filaments and actin elongation factors to control the rate and extent of polymerization. Nucleation and elongation factors identified to date include Arp2/3 complex, formins, Ena/VASP, and newcomers Spire, Cobl, and Lmod. Here, we discuss recent advances in understanding their activities and mechanisms and new evidence for their cooperation and interaction in vivo. Earlier models had suggested that different nucleators function independently to assemble distinct actin arrays. However, more recent observations indicate that the construction of most cellular actin networks depends on the activities of multiple actin assembly-promoting factors working in concert.

  16. The busiest of all ribosomal assistants: elongation factor Tu.

    PubMed

    Kavaliauskas, Darius; Nissen, Poul; Knudsen, Charlotte R

    2012-04-01

    During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, elongation, and termination. After a protein has been synthesized, the components of the translation apparatus are recycled. During each phase of translation, the ribosome collaborates with specific translation factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the elongation step is repeated a large number of times, corresponding to the number of amino acids constituting the protein of interest. In bacteria, elongation factor Tu plays a central role during the selection of the correct amino acids throughout the elongation phase of translation. Elongation factor Tu is the main subject of this review. PMID:22409271

  17. Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

    PubMed

    Knight, John R P; Bastide, Amandine; Roobol, Anne; Roobol, Jo; Jackson, Thomas J; Utami, Wahyu; Barrett, David A; Smales, C Mark; Willis, Anne E

    2015-01-15

    Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

  18. Catalytic effects of elongation factor Ts on polypeptide synthesis

    PubMed Central

    Ruusala, Tarmo; Ehrenberg, Måns; Kurland, Charles G.

    1982-01-01

    The kinetic parameters which characterize the interaction between elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) have been determined in a poly(uridylic acid)-primed translation system. The EF-Ts catalyzed release of GDP from EF-Tu was measured independently in a nucleotide exchange assay. We conclude that the rate-limiting step for the EF-Tu cycle in protein synthesis in the absence of EF-Ts is the release of GDP. By adding EF-Ts the time of this step is reduced from 90 s to 30 ms. Half maximal rate is obtained at an EF-Ts concentration of 2.5 x 10−6 M. PMID:16453409

  19. Maintenance of Transcription-Translation Coupling by Elongation Factor P

    PubMed Central

    Elgamal, Sara

    2016-01-01

    ABSTRACT Under conditions of tight coupling between translation and transcription, the ribosome enables synthesis of full-length mRNAs by preventing both formation of intrinsic terminator hairpins and loading of the transcription termination factor Rho. While previous studies have focused on transcription factors, we investigated the role of Escherichia coli elongation factor P (EF-P), an elongation factor required for efficient translation of mRNAs containing consecutive proline codons, in maintaining coupled translation and transcription. In the absence of EF-P, the presence of Rho utilization (rut) sites led to an ~30-fold decrease in translation of polyproline-encoding mRNAs. Coexpression of the Rho inhibitor Psu fully restored translation. EF-P was also shown to inhibit premature termination during synthesis and translation of mRNAs encoding intrinsic terminators. The effects of EF-P loss on expression of polyproline mRNAs were augmented by a substitution in RNA polymerase that accelerates transcription. Analyses of previously reported ribosome profiling and global proteomic data identified several candidate gene clusters where EF-P could act to prevent premature transcription termination. In vivo probing allowed detection of some predicted premature termination products in the absence of EF-P. Our findings support a model in which EF-P maintains coupling of translation and transcription by decreasing ribosome stalling at polyproline motifs. Other regulators that facilitate ribosome translocation through roadblocks to prevent premature transcription termination upon uncoupling remain to be identified. PMID:27624127

  20. Engineering the elongation factor Tu for efficient selenoprotein synthesis

    PubMed Central

    Haruna, Ken-ichi; Alkazemi, Muhammad H.; Liu, Yuchen; Söll, Dieter; Englert, Markus

    2014-01-01

    Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically—using the UAG amber codon—inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O6-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production. PMID:25064855

  1. Actin filament nucleation and elongation factors--structure-function relationships.

    PubMed

    Dominguez, Roberto

    2009-01-01

    The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tbeta4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been "outsourced" to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.

  2. Gain and loss of elongation factor genes in green algae

    PubMed Central

    Cocquyt, Ellen; Verbruggen, Heroen; Leliaert, Frederik; Zechman, Frederick W; Sabbe, Koen; De Clerck, Olivier

    2009-01-01

    Background Two key genes of the translational apparatus, elongation factor-1 alpha (EF-1α) and elongation factor-like (EFL) have an almost mutually exclusive distribution in eukaryotes. In the green plant lineage, the Chlorophyta encode EFL except Acetabularia where EF-1α is found, and the Streptophyta possess EF-1α except Mesostigma, which has EFL. These results raise questions about evolutionary patterns of gain and loss of EF-1α and EFL. A previous study launched the hypothesis that EF-1α was the primitive state and that EFL was gained once in the ancestor of the green plants, followed by differential loss of EF-1α or EFL in the principal clades of the Viridiplantae. In order to gain more insight in the distribution of EF-1α and EFL in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time Markov models. Results Within the Chlorophyta, EF-1α is shown to be present in three ulvophycean orders (i.e., Dasycladales, Bryopsidales, Siphonocladales) and the genus Ignatius. Models describing gene gain-loss dynamics revealed that the presence of EF-1α, EFL or both genes along the backbone of the green plant phylogeny is highly uncertain due to sensitivity to branch lengths and lack of prior knowledge about ancestral states or rates of gene gain and loss. Model refinements based on insights gained from the EF-1α phylogeny reduce uncertainty but still imply several equally likely possibilities: a primitive EF-1α state with multiple independent EFL gains or coexistence of both genes in the ancestor of the Viridiplantae or Chlorophyta followed by differential loss of one or the other gene in the various lineages. Conclusion EF-1α is much more common among green algae than previously thought. The mutually exclusive distribution of EF-1α and EFL is confirmed in a large sample of green plants. Hypotheses about the gain

  3. Mammalian elongation factor 4 regulates mitochondrial translation essential for spermatogenesis.

    PubMed

    Gao, Yanyan; Bai, Xiufeng; Zhang, Dejiu; Han, Chunsheng; Yuan, Jing; Liu, Wenbin; Cao, Xintao; Chen, Zilei; Shangguan, Fugen; Zhu, Zhenyuan; Gao, Fei; Qin, Yan

    2016-05-01

    Elongation factor 4 (EF4) is a key quality-control factor in translation. Despite its high conservation throughout evolution, EF4 deletion in various organisms has not yielded a distinct phenotype. Here we report that genetic ablation of mitochondrial EF4 (mtEF4) in mice causes testis-specific dysfunction in oxidative phosphorylation, leading to male infertility. Deletion of mtEF4 accelerated mitochondrial translation at the cost of producing unstable proteins. Somatic tissues overcame this defect by activating mechanistic (mammalian) target of rapamycin (mTOR), thereby increasing rates of cytoplasmic translation to match rates of mitochondrial translation. However, in spermatogenic cells, the mTOR pathway was downregulated as part of the developmental program, and the resulting inability to compensate for accelerated mitochondrial translation caused cell-cycle arrest and apoptosis. We detected the same phenotype and molecular defects in germline-specific mtEF4-knockout mice. Thus, our study demonstrates cross-talk between mtEF4-dependent quality control in mitochondria and cytoplasmic mTOR signaling.

  4. The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation*

    PubMed Central

    Tavares, Clint D. J.; Ferguson, Scarlett B.; Giles, David H.; Wang, Qiantao; Wellmann, Rebecca M.; O'Brien, John P.; Warthaka, Mangalika; Brodbelt, Jennifer S.; Ren, Pengyu; Dalby, Kevin N.

    2014-01-01

    Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca2+/CaM binds eEF-2K with high affinity (Kd(CaM)app = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 104-fold (kauto = 2.6 ± 0.3 s−1). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca2+/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing kcatapp for peptide substrate phosphorylation by 103-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (kcatapp/Km(Pep-S)app), with equal contributions from kcatapp and Km(Pep-S)app, suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output. PMID:25012662

  5. Movement of elongation factor G between compact and extended conformations.

    PubMed

    Salsi, Enea; Farah, Elie; Netter, Zoe; Dann, Jillian; Ermolenko, Dmitri N

    2015-01-30

    Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer. Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pretranslocation ribosomes or with posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to but likely precedes both GTP hydrolysis and mRNA/tRNA translocation.

  6. Movement of Elongation Factor G between Compact and Extended Conformations

    PubMed Central

    Salsi, Enea; Farah, Elie; Netter, Zoe; Dann, Jillian; Ermolenko, Dmitri N.

    2014-01-01

    Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer (smFRET). Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pre- or posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to, but likely precedes both GTP hydrolysis and mRNA/tRNA translocation. PMID:25463439

  7. Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

    PubMed Central

    Hersch, Steven J.; Roy, Hervé; Wiggers, J. Brad; Leung, Andrea S.; Buranyi, Stephen; Xie, Jinglin Lucy; Dare, Kiley; Ibba, Michael; Navarre, William Wiley

    2012-01-01

    Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin. PMID:22081389

  8. Convergent intron gains in hymenopteran elongation factor-1α.

    PubMed

    Klopfstein, Seraina; Ronquist, Fredrik

    2013-04-01

    The eukaryotic translation elongation factor-1α gene (eEF1A) has been used extensively in higher level phylogenetics of insects and other groups, despite being present in two or more copies in several taxa. Orthology assessment has relied heavily on the position of introns, but the basic assumption of low rates of intron loss and absence of convergent intron gains has not been tested thoroughly. Here, we study the evolution of eEF1A based on a broad sample of taxa in the insect order Hymenoptera. The gene is universally present in two copies - F1 and F2 - both of which apparently originated before the emergence of the order. An elevated ratio of non-synonymous versus synonymous substitutions and differences in rates of amino acid replacements between the copies suggest that they evolve independently, and phylogenetic methods clearly cluster the copies separately. The F2 copy appears to be ancient; it is orthologous with the copy known as F1 in Diptera, and is likely present in most insect orders. The hymenopteran F1 copy, which may or may not be unique to this order, apparently originated through retroposition and was originally intron free. During the evolution of the Hymenoptera, it has successively accumulated introns, at least three of which have appeared at the same position as introns in the F2 copy or in eEF1A copies in other insects. The sites of convergent intron gain are characterized by highly conserved nucleotides that strongly resemble specific intron-associated sequence motifs, so-called proto-splice sites. The significant rate of convergent intron gain renders intron-exon structure unreliable as an indicator of orthology in eEF1A, and probably also in other protein-coding genes.

  9. The interaction between bacterial transcription factors and RNA polymerase during the transition from initiation to elongation.

    PubMed

    Yang, Xiao; Lewis, Peter J

    2010-01-01

    There are three stages of transcription: initiation, elongation and termination, and traditionally there has been a clear distinction between the stages. The specificity factor sigma is completely released from bacterial RNA polymerase after initiation, and then recycled for another round of transcription. Elongation factors then associate with the polymerase followed by termination factors (where necessary). These factors dissociate prior to initiation of a new round of transcription. However, there is growing evidence suggesting that sigma factors can be retained in the elongation complex. The structure of bacterial RNAP in complex with an essential elongation factor NusA has recently been published, which suggested rather than competing for the major σ binding site, NusA binds to a discrete region on RNAP. A model was proposed to help explain the way in which both factors could be associated with RNAP during the transition from transcription initiation to elongation.

  10. Pleiohomeotic Interacts with the Core Transcription Elongation Factor Spt5 to Regulate Gene Expression in Drosophila

    PubMed Central

    Jennings, Barbara H.

    2013-01-01

    The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. Here we demonstrate that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. Our results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner. PMID:23894613

  11. Modes of Action of ADP-Ribosylated Elongation Factor 2 in Inhibiting the Polypeptide Elongation Cycle: A Modeling Study

    PubMed Central

    Chen, Kevin C.; Xie, Honglin; Cai, Yujie

    2013-01-01

    Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2) leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2) causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome. PMID:23861744

  12. Repression of In Vivo Synthesis of the Mitochondrial Elongation Factors T and G in Saccharomyces fragilis

    PubMed Central

    Richter, D.

    1973-01-01

    In vivo synthesis of the mitochondrial elongation factors T and G in the yeast Saccharomyces fragilis can be repressed. Enzymatic activity assays and immunochemical titration methods reveal that cells grown in the presence of 8% glucose or in the absence of oxygen contain relatively lower amounts of mitochondrial elongation factors than cells grown in the presence of lactate. In contrast, in vivo production of the cytoplasmic elongation factors 1 and 2 does not respond to such a change of extracellular conditions. The rate of growth does not affect the level of the mitochondrial elongation factors. Production of both enzymes is almost constant during logarithmic growth, but decreases when the stationary phase is reached. Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not block but, rather, seems to enhance the in vivo synthesis of mitochondrial T or G. PMID:4717524

  13. Interaction of elongation factor 1 with aminoacylated brome mosaic virus and tRNA's.

    PubMed Central

    Bastin, M; Hall, T C

    1976-01-01

    Tyrosylated Brome mosaic virus RNA was found to interact with a binary complex of wheat germ, elongation factor 1 and [3H]GTP. Increasing amounts of the aminoacylated viral RNA proportionately reduced radioactivity bound to a nitrocellulose filter, as has previously been noted by others for the charged forms of tobacco mosaic virus, turnip yellow mosaic virus, and tRNA's. However, Sephadex chromatography of the products showed that instead of forming the ternary complex elongation factor-GTP-aminoacyl RNA, the viral RNA caused release of GTP from its complex with elongation factor. Acetylated tyrosyl Brome mosaic virus RNA did not react with the binary complex,and only a slight degree, if any, of stabilization of tyrosine bound to viral RNA was observed after interaction with elongation factor 1. Although such interactions are similar to the reaction of elongation factor with aminoacyl-tRNA , the release of GTP is different and accentuates the possible role for aminoacylation in transcription rather than in translation events. PMID:978788

  14. A putative transcriptional elongation factor hIws1 is essential for mammalian cell proliferation

    SciTech Connect

    Liu Zhangguo; Zhou Zhongwei; Chen Guohong; Bao Shilai . E-mail: slbao@genetics.ac.cn

    2007-02-02

    Iws1 has been implicated in transcriptional elongation by interaction with RNA polymerase II (RNAP II) and elongation factor Spt6 in budding yeast Saccharomyces cerevisiae, and association with transcription factor TFIIS in mammalian cells, but its role in controlling cell growth and proliferation remains unknown. Here we report that the human homolog of Iws1, hIws1, physically interacts with protein arginine methyltransferases PRMT5 which methylates elongation factor Spt5 and regulates its interaction with RNA polymerase II. Gene-specific silencing of hIws1 by RNA interference reveals that hIws1 is essential for cell viability. GFP fusion protein expression approaches demonstrate that the hIws1 protein is located in the nucleus, subsequently, two regions harbored within the hIws1 protein are demonstrated to contain nuclear localization signals (NLSs). In addition, mouse homolog of hiws1 is found to express ubiquitously in various tissues.

  15. Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

    PubMed

    Krastel, Philipp; Roggo, Silvio; Schirle, Markus; Ross, Nathan T; Perruccio, Francesca; Aspesi, Peter; Aust, Thomas; Buntin, Kathrin; Estoppey, David; Liechty, Brigitta; Mapa, Felipa; Memmert, Klaus; Miller, Howard; Pan, Xuewen; Riedl, Ralph; Thibaut, Christian; Thomas, Jason; Wagner, Trixie; Weber, Eric; Xie, Xiaobing; Schmitt, Esther K; Hoepfner, Dominic

    2015-08-24

    Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1. PMID:26179970

  16. Transcription factors Sox10 and Sox2 functionally interact with positive transcription elongation factor b in Schwann cells.

    PubMed

    Arter, Juliane; Wegner, Michael

    2015-02-01

    Sox proteins are mechanistically versatile regulators with established relevance to different developmental processes and crucial impact on chromatin structure, DNA conformation, and transcriptional initiation. Here, we show that Sox2 and Sox10, two Sox proteins important for Schwann cell development, also have the capability to activate transcriptional elongation in a Schwann cell line by recruiting the positive transcription elongation factor b. Recruitment is mediated by physical interaction between the carboxyterminal transactivation domains of the two Sox proteins and the Cyclin T1 subunit of positive transcription elongation factor b, with interaction interfaces for the two Sox proteins being mapped to adjacent regions of the central part of Cyclin T1. Supporting the relevance of this interaction to Schwann cell development, transcription of myelin genes appears regulated at the level of elongation. Our results thus add a new facet to the activity of Sox proteins and expand the functional repertoire of this important group of developmental regulators. Sox transcription factors are important regulators of nervous system development. While they are known to regulate transcription by recruiting and stabilizing the RNA polymerase II preinitiation complex directly or with help of the Mediator complex, this study provides evidence that Sox10 and Sox2 additionally influence transcription in glial cells at the elongation stage by recruiting P-TEFb. Cdk9, cyclin-dependent kinase 9; P-TEFb, positive transcription elongation factor b; Pol II, RNA polymerase II; Sox, Sox2 or Sox10 protein.

  17. SHORT HYPOCOTYL1 Encodes a SMARCA3-Like Chromatin Remodeling Factor Regulating Elongation1[OPEN

    PubMed Central

    Bo, Kailiang; Behera, Tusar K.; Pandey, Sudhakar; Wen, Changlong; Wang, Yuhui; Simon, Philipp W.; Li, Yuhong

    2016-01-01

    In Arabidopsis (Arabidopsis thaliana), the UVR8-mediated signaling pathway is employed to attain UVB protection and acclimation to deal with low-dosage UVB (LDUVB)-induced stresses. Here, we identified SHORT HYPOCOTYL1 (SH1) in cucumber (Cucumis sativus), which regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway. We showed that hypocotyl elongation in cucumbers carrying the recessive sh1 allele was LDUVB insensitive and that Sh1 encoded a human SMARCA3-like chromatin remodeling factor. The allele frequency and distribution pattern at this locus among natural populations supported the wild cucumber origin of sh1 for local adaptation, which was under selection during domestication. The cultivated cucumber carries predominantly the Sh1 allele; the sh1 allele is nearly fixed in the semiwild Xishuangbanna cucumber, and the wild cucumber population is largely at Hardy-Weinberg equilibrium for the two alleles. The SH1 protein sequence was highly conserved among eukaryotic organisms, but its regulation of hypocotyl elongation in cucumber seems to be a novel function. While Sh1 expression was inhibited by LDUVB, its transcript abundance was highly correlated with hypocotyl elongation rate and the expression level of cell-elongation-related genes. Expression profiling of key regulators in the UVR8 signaling pathway revealed significant differential expression of CsHY5 between two near isogenic lines of Sh1. Sh1 and CsHY5 acted antagonistically at transcriptional level. A working model was proposed in which Sh1 regulates LDUVB-dependent hypocotyl elongation in cucumber through changing the chromatin states and thus the accessibility of CsHY5 in the UVR8 signaling pathway to promoters of LDUVB-responsive genes for hypocotyl elongation. PMID:27559036

  18. Heat-induced Accumulation of Chloroplast Protein Synthesis Elongation Factor, EF-TU, in Winter Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays L.). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum L.). In this ...

  19. Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

    PubMed Central

    González-Terán, Bárbara; Cortés, José R.; Manieri, Elisa; Matesanz, Nuria; Verdugo, ρngeles; Rodríguez, María E.; González-Rodríguez, ρgueda; Valverde, ρngela; Martín, Pilar; Davis, Roger J.; Sabio, Guadalupe

    2012-01-01

    Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-α. TNF-α is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-α expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38γ/δ MAPK proteins is required for the elongation of nascent TNF-α protein in macrophages. The MKK3/6-p38γ/δ pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-α elongation. These results identify a new signaling pathway that regulates TNF-α production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-α production is involved. PMID:23202732

  20. Membrane elongation factors in organelle maintenance: the case of peroxisome proliferation

    PubMed Central

    Koch, Johannes; Brocard, Cécile

    2011-01-01

    Separation of metabolic pathways in organelles is critical for eukaryotic life. Accordingly, the number, morphology and function of organelles have to be maintained through processes linked with membrane remodeling events. Despite their acknowledged significance and intense study many questions remain about the molecular mechanisms by which organellar membranes proliferate. Here, using the example of peroxisome proliferation, we give an overview of how proteins elongate membranes. Subsequent membrane fission is achieved by dynamin-related proteins shared with mitochondria. We discuss basic criteria that membranes have to fulfill for these fission factors to complete the scission. Because peroxisome elongation is always associated with unequal distribution of matrix and membrane proteins, we propose peroxisomal division to be non-stochastic and asymmetric. We further show that these organelles need not be functional to carry on membrane elongation and present the most recent findings concerning members of the Pex11 protein family as membrane elongation factors. These factors, beside known proteins such as BAR-domain proteins, represent another family of proteins containing an amphipathic α-helix with membrane bending activity. PMID:21984887

  1. Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

    PubMed

    McKnight, Robert A; Yost, Christian C; Zinkhan, Erin K; Fu, Qi; Callaway, Christopher W; Fung, Camille M

    2016-08-01

    Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription. PMID:27317589

  2. Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

    PubMed

    Suzuki, Atsuo; Miyawaki, Yuhri; Okuyama, Eriko; Murata, Moe; Ando, Yumi; Kato, Io; Takagi, Yuki; Takagi, Akira; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2013-01-01

    Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

  3. Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

    PubMed Central

    Archambault, J; Lacroute, F; Ruet, A; Friesen, J D

    1992-01-01

    Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII. Images PMID:1508210

  4. Synthesis of reinitiated transcripts by mammalian RNA polymerase II is controlled by elongation factor SII.

    PubMed Central

    Szentirmay, M N; Sawadogo, M

    1993-01-01

    Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes. Images PMID:8223477

  5. Phosphorylation of elongation factor 2 during Ca(2+)-mediated secretion from rat parotid acini.

    PubMed Central

    Hincke, M T; Nairn, A C

    1992-01-01

    In this paper we report the rapid phosphorylation of a cytosolic 100 kDa protein during stimulation of secretion from dispersed aggregates of parotid acinar cells with Ca(2+)-mobilizing secretagogues (carbachol, Substance P, ATP and the Ca2+ ionophore A23187). Phosphorylation was inhibited by removal of extracellular Ca2+ but was not observed during stimulation with phorbol esters, suggesting that this protein is not a substrate for protein kinase C. Two-dimensional PAGE and immunoprecipitation with a specific antiserum indicated that this protein is elongation factor 2, whose Ca2+ calmodulin-dependent phosphorylation has been shown to inhibit protein synthesis [Nairn & Palfrey (1987) J. Biol. Chem. 262, 17299-17303]. These results suggest that phosphorylation of elongation factor 2 is the molecular mechanism for the inhibition of protein synthesis which has been previously observed in rat parotid cells during stimulation with Ca(2+)-mobilizing secretagogues. Images Fig. 1. Fig. 3. Fig. 5. Fig. 6. PMID:1372803

  6. The in vivo dynamics of TCERG1, a factor that couples transcriptional elongation with splicing.

    PubMed

    Sánchez-Hernández, Noemí; Boireau, Stéphanie; Schmidt, Ute; Muñoz-Cobo, Juan Pablo; Hernández-Munain, Cristina; Bertrand, Edouard; Suñé, Carlos

    2016-04-01

    Coupling between transcription and RNA processing is key for gene regulation. Using live-cell photobleaching techniques, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing. We demonstrate that TCERG1 is highly mobile in the nucleoplasm and that this mobility is slightly decreased when it is associated with speckles. Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) but not α-amanitin treatment reduced the mobility of TCERG1, which suggests interaction with paused transcription elongation complexes. We found that TCERG1 mobility is rapid at the transcription site (TS) of a reporter that splices post-transcriptionally and that TCERG1 is recruited to the active TS independent of the CTD of RNAPII, thus excluding phosphorylated CTD as a requirement for recruiting this factor to the TS. Importantly, the mobility of TCERG1 is reduced when the reporter splices cotranscriptionally, which suggests that TCERG1 forms new macromolecular complexes when splicing occurs cotranscriptionally. In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation. Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes. This finding has conceptual implications for understanding the coupling between transcription and RNA processing. PMID:26873599

  7. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling.

    PubMed

    Zhou, Xin; Zhang, Zhong-Lin; Park, Jeongmoo; Tyler, Ludmila; Yusuke, Jikumaru; Qiu, Kai; Nam, Edward A; Lumba, Shelley; Desveaux, Darrell; McCourt, Peter; Kamiya, Yuji; Sun, Tai-Ping

    2016-08-01

    The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways. PMID:27255484

  8. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling1[OPEN

    PubMed Central

    Zhou, Xin; Zhang, Zhong-Lin; Tyler, Ludmila; Yusuke, Jikumaru; Qiu, Kai; Lumba, Shelley; Desveaux, Darrell; McCourt, Peter; Sun, Tai-ping

    2016-01-01

    The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways. PMID:27255484

  9. Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing.

    PubMed

    Kudlicki, W; Coffman, A; Kramer, G; Hardesty, B

    1997-12-19

    The translation elongation factor (EF) Tu has chaperone-like capacity to promote renaturation of denatured rhodanese. This renaturation activity is greatly increased under conditions in which the factor can oscillate between the open and closed conformations that are induced by GDP and GTP, respectively. Oscillation occurs during GTP hydrolysis and subsequent replacement of GDP by EF-Ts which is then displaced by GTP. Renaturation of rhodanese and GTP hydrolysis by EF-Tu are greatly enhanced by the guanine nucleotide exchange factor EF-Ts. However, renaturation is reduced under conditions that stabilize EF-Tu in either the open or closed conformation. Both GDP and the nonhydrolyzable analog of GTP, GMP-PCP, inhibit renaturation. Kirromycin and pulvomycin, antibiotics that specifically bind to EF-Tu and inhibit its activity in peptide elongation, also strongly inhibit EF-Tu-mediated renaturation of denatured rhodanese to levels near those observed for spontaneous, unassisted refolding. Kirromycin locks EF-Tu in the open conformation in the presence of either GTP or GDP, whereas pulvomycin locks the factor in the closed conformation. The results lead to the conclusion that flexing of EF-Tu, especially as occurs between its open and closed conformations, is a major factor in its chaperone-like refolding activity.

  10. Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3

    PubMed Central

    Shan, Chun-Min; Shangguan, Xiao-Xia; Zhao, Bo; Zhang, Xiu-Fang; Chao, Lu-men; Yang, Chang-Qing; Wang, Ling-Jian; Zhu, Hua-Yu; Zeng, Yan-Da; Guo, Wang-Zhen; Zhou, Bao-Liang; Hu, Guan-Jing; Guan, Xue-Ying; Chen, Z. Jeffrey; Wendel, Jonathan F.; Zhang, Tian-Zhen; Chen, Xiao-Ya

    2014-01-01

    Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3–GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation. PMID:25413731

  11. Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells.

    PubMed

    Fujinaga, Koh; Luo, Zeping; Schaufele, Fred; Peterlin, B Matija

    2015-01-16

    Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation. However, no good method exists to analyze this P-TEFb equilibrium in living cells. Only inaccurate and labor-intensive cell-free biochemical assays are currently available. In this study, we present the first experimental system to monitor P-TEFb activation in living cells. We created a bimolecular fluorescence complementation assay to detect interactions between P-TEFb and its substrate, the C-terminal domain of RNA polymerase II. When cells were treated with suberoylanilide hydroxamic acid, which releases P-TEFb from the 7SK small nuclear ribonucleoprotein, they turned green. Other known P-TEFb-releasing agents, including histone deacetylase inhibitors, bromodomain and extraterminal bromodomain inhibitors, and protein kinase C agonists, also scored positive in this assay. Finally, we identified 5'-azacytidine as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with activation of human HIV and HEXIM1 transcription. Thus, our visualization of P-TEFb activation by fluorescent complementation assay could be used to find new P-TEFb-releasing agents, compare different classes of agents, and assess their efficacy singly and/or in combination.

  12. Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

    PubMed Central

    Moore, Claire E. J.; Mikolajek, Halina; Regufe da Mota, Sergio; Wang, Xuemin; Kenney, Justin W.; Werner, Jörn M.

    2015-01-01

    Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors. PMID:25755286

  13. Direct phylogenetic evidence for lateral transfer of elongation factor-like gene

    PubMed Central

    Kamikawa, Ryoma; Inagaki, Yuji; Sako, Yoshihiko

    2008-01-01

    Genes encoding elongation factor-like (EFL) proteins, which show high similarity to elongation factor-1α (EF-1α), have been found in phylogenetically distantly related eukaryotes. The sporadic distribution of “EFL-containing” lineages within “EF-1α-containing” lineages indirectly, but strongly, suggests lateral gene transfer as the principal driving force in EFL evolution. However, one of the most critical aspects in the above hypothesis, the donor lineages in any putative cases of lateral EFL gene transfer, remained unclear. In this study, we provide direct evidence for lateral transfer of an EFL gene through the analyses of 10 diatom EFL genes. All diatom EFL homologues tightly clustered in phylogenetic analyses, suggesting acquisition of the exogenous EFL gene early in diatom evolution. Our survey additionally identified Thalassiosira pseudonana as a eukaryote bearing EF-1α and EFL genes and secondary EFL gene loss in Phaeodactylum tricornutum, the complete genome of which encodes only the EF-1α gene. Most importantly, the EFL phylogeny recovered a robust grouping of homologues from diatoms, the cercozoan Bigelowiella natans, and the foraminifer Planoglabratella opecularis, with the diatoms nested within the Bigelowiella plus Planoglabratella (Rhizaria) grouping. The particular relationships recovered are further consistent with two characteristic sequence motifs. The best explanation of our data analyses is an EFL gene transfer from a foraminifer to a diatom, the first case in which the donor–recipient relationship was clarified. Finally, based on a reverse transcriptase quantitative PCR assay and the genome information of Thalassiosira and Phaeodactylum, we propose the loss of elongation factor function in Thalassiosira EF-1α. PMID:18458344

  14. Distinct Clinical Phenotypes Associated with a Mutation in the Mitochondrial Translation Elongation Factor EFTs

    PubMed Central

    Smeitink, Jan A. M.; Elpeleg, Orly; Antonicka, Hana; Diepstra, Heleen; Saada, Ann; Smits, Paulien; Sasarman, Florin; Vriend, Gert; Jacob-Hirsch, Jasmine; Shaag, Avraham; Rechavi, Gideon; Welling, Brigitte; Horst, Jürgen; Rodenburg, Richard J.; van den Heuvel, Bert; Shoubridge, Eric A.

    2006-01-01

    The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here, we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60%, respectively, and the amounts of assembled complexes I, IV, and V were reduced by 35%–91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus. PMID:17033963

  15. Resveratrol Regulates Pathologic Angiogenesis by a Eukaryotic Elongation Factor-2 Kinase-Regulated Pathway

    PubMed Central

    Khan, Aslam A.; Dace, Dru S.; Ryazanov, Alexey G.; Kelly, Jennifer; Apte, Rajendra S.

    2010-01-01

    Abnormal angiogenesis is central to the pathophysiology of diverse disease processes including cancers, ischemic and atherosclerotic heart disease, and visually debilitating eye disease. Resveratrol is a naturally occurring phytoalexin that has been demonstrated to ameliorate and decelerate the aging process as well as blunt end organ damage from obesity. These effects of resveratrol are largely mediated by members of the sirtuin family of proteins. We demonstrate that resveratrol can inhibit pathological angiogenesis in vivo and in vitro by a sirtuin-independent pathway. Resveratrol inhibits the proliferation and migration of vascular endothelial cells by activating eukaryotic elongation factor-2 kinase. The active kinase in turn phosphorylates and inactivates elongation factor-2, a key mediator of ribosomal transfer and protein translation. Functional inhibition of the kinase by gene deletion in vivo or RNA as well as pharmacological inhibition in vitro is able to completely reverse the effects of resveratrol on blood vessel growth. These studies have identified a novel and critical pathway that promotes aberrant vascular proliferation and one that is amenable to modulation by pharmacological means. In addition, these results have uncovered a sirtuin-independent pathway by which resveratrol regulates angiogenesis. PMID:20472894

  16. Transcription termination factor rho prefers catalytically active elongation complexes for releasing RNA.

    PubMed

    Dutta, Dipak; Chalissery, Jisha; Sen, Ranjan

    2008-07-18

    RNA polymerase pauses at different DNA sequences during transcription elongation, and this pausing is associated with distinct conformational state(s) of the elongation complex (EC). Transcription termination by the termination factor Rho, an RNA-dependent molecular motor, requires pausing of the EC in the termination zone of Rho-dependent terminators. We hypothesized that the conformational state(s) of the EC associated with this pausing would influence the action of Rho. Analyses of the pausing behavior of the EC at the termination points of two well known Rho-dependent terminators revealed that Rho prefers actively transcribing complexes for termination. RNA release kinetics from stalled ECs showed that the rate of RNA release by Rho was reduced if the EC was irreversibly backtracked, if its RNA exit channel was modified by an RNA hairpin, or the bridge helix/trigger loop movement in its active site was perturbed. These defects were overcome significantly by enhancing the rate of ATP hydrolysis either by increasing the concentration of ATP or by using a Rho mutant with higher ATPase activity. We propose that the force generated from ATP hydrolysis of Rho is the key factor in dislodging the EC through its molecular motor action, and this process is facilitated when the EC is in a catalytically competent state, undergoing rapid "Brownian ratchet" motion.

  17. Arginine-rhamnosylation as new strategy to activate translation elongation factor P.

    PubMed

    Lassak, Jürgen; Keilhauer, Eva C; Fürst, Maximilian; Wuichet, Kristin; Gödeke, Julia; Starosta, Agata L; Chen, Jhong-Min; Søgaard-Andersen, Lotte; Rohr, Jürgen; Wilson, Daniel N; Häussler, Susanne; Mann, Matthias; Jung, Kirsten

    2015-04-01

    Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development. PMID:25686373

  18. Identification of two genes coding for the translation elongation factor EF-1 alpha of S. cerevisiae.

    PubMed

    Schirmaier, F; Philippsen, P

    1984-12-20

    The translation elongation factor EF-1 alpha of the yeast Saccharomyces cerevisiae is coded for by two genes, called TEF1 and TEF2. Both genes were cloned. TEF1 maps on chromosome II close to LYS2. The location of TEF2 is unknown. TEF2 alone is sufficient to promote growth of the cells as shown with a strain deleted for TEF1. TEF1 and TEF2 were originally identified as two strongly transcribed genes, which most likely code for an identical or nearly identical protein as judged from S1 nuclease protection experiments with mRNA-DNA hybrids. The DNA sequence analysis of TEF1 allowed the prediction of the protein sequence. This was shown, by a search in the Dayhoff protein data bank, to represent the translation elongation factor EF-1 alpha due to the striking similarity to EF-1 alpha from the shrimp Artemia. A search for TEF1 homologous sequences in several yeast species shows, in most cases, duplicated genes and a much higher sequence conservation than among genes encoding amino acid biosynthetic enzymes. PMID:6396088

  19. Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

    PubMed Central

    Li, Wen; Liu, Zheng; Koripella, Ravi Kiran; Langlois, Robert; Sanyal, Suparna; Frank, Joachim

    2015-01-01

    During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome. PMID:26229983

  20. Identification and quantitation of elongation factor EF-P in Escherichia coli cell-free extracts.

    PubMed

    An, G; Glick, B R; Friesen, J D; Ganoza, M C

    1980-11-01

    EF-P, an elongation factor that stimulates peptide bond synthesis in vitro with some aminoacyl-tRNAs, has been identified by two-dimensional gel electrophoresis and the cellular content at three points in the growth curve has been measured. The molecular weight of EF-P is estimated to be 21 000. EF-P is a slightly acidic protein whose isoelectric point is close to RNA polymerase subunit alpha. The amount of EF-P present in Escherichia coli is about 1/10th that of EF-G and the level is independent of the stage of cell growth; there is about one EF-P per 10 ribosomes. It is also shown that a highly purified preparation of EF-P is free of all known protein synthesis factors and ribosomal proteins. PMID:7011506

  1. Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.

    PubMed

    van Damme, H T; Amons, R; Möller, W

    1992-08-01

    In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced. PMID:1499548

  2. Evolution of eukaryotic translation elongation and termination factors: variations of evolutionary rate and genetic code deviations.

    PubMed

    Moreira, David; Kervestin, Stéphanie; Jean-Jean, Olivier; Philippe, Hervé

    2002-02-01

    Translation is carried out by the ribosome and several associated protein factors through three consecutive steps: initiation, elongation, and termination. Termination remains the least understood of them, partly because of the nonuniversality of the factors involved. To get some insights on the evolution of eukaryotic translation termination, we have compared the phylogeny of the release factors eRF1 and eRF3 to that of the elongation factors EF-1alpha and EF-2, with special focus on ciliates. Our results show that these four translation proteins have experienced different modes of evolution. This is especially evident for the EF-1alpha, EF-2, and eRF1 ciliate sequences. Ciliates appear as monophyletic in the EF-2 phylogenetic tree but not in the EF-1alpha and eRF1 phylogenetic trees. This seems to be mainly because of phylogeny reconstruction artifacts (the long-branch attraction) produced by the acceleration of evolutionary rate of ciliate EF-1alpha and eRF1 sequences. Interaction with the highly divergent actin found in ciliates, or on the contrary, loss of interaction, could explain the acceleration of the evolutionary rate of the EF-1alpha sequences. In the case of ciliate eRF1 sequences, their unusually high evolutionary rate may be related to the deviations in the genetic code usage found in diverse ciliates. These deviations involve a relaxation (or even abolition) of the recognition of one or two stop codons by eRF1. To achieve this, structural changes in eRF1 are needed, and this may affect its evolutionary rate. Eukaryotic translation seems to have followed a mosaic evolution, with its different elements governed by different selective pressures. However, a correlation analysis shows that, beneath the disagreement shown by the different translation proteins, their concerted evolution can still be made apparent when they are compared with other proteins that are not involved in translation.

  3. The elongation factor Spt5 facilitates transcription initiation for rapid induction of inflammatory-response genes

    PubMed Central

    Diamant, Gil; Bahat, Anat; Dikstein, Rivka

    2016-01-01

    A subset of inflammatory-response NF-κB target genes is activated immediately following pro-inflammatory signal. Here we followed the kinetics of primary transcript accumulation after NF-κB activation when the elongation factor Spt5 is knocked down. While elongation rate is unchanged, the transcript synthesis at the 5′-end and at the earliest time points is delayed and reduced, suggesting an unexpected role in early transcription. Investigating the underlying mechanism reveals that the induced TFIID–promoter association is practically abolished by Spt5 depletion. This effect is associated with a decrease in promoter-proximal H3K4me3 and H4K5Ac histone modifications that are differentially required for rapid transcriptional induction. In contrast, the displacement of TFIIE and Mediator, which occurs during promoter escape, is attenuated in the absence of Spt5. Our findings are consistent with a central role of Spt5 in maintenance of TFIID–promoter association and promoter escape to support rapid transcriptional induction and re-initiation of inflammatory-response genes. PMID:27180651

  4. Elongation Factor-Tu (EF-Tu) proteins structural stability and bioinformatics in ancestral gene reconstruction

    NASA Astrophysics Data System (ADS)

    Dehipawala, Sunil; Nguyen, A.; Tremberger, G.; Cheung, E.; Schneider, P.; Lieberman, D.; Holden, T.; Cheung, T.

    2013-09-01

    A paleo-experimental evolution report on elongation factor EF-Tu structural stability results has provided an opportunity to rewind the tape of life using the ancestral protein sequence reconstruction modeling approach; consistent with the book of life dogma in current biology and being an important component in the astrobiology community. Fractal dimension via the Higuchi fractal method and Shannon entropy of the DNA sequence classification could be used in a diagram that serves as a simple summary. Results from biomedical gene research provide examples on the diagram methodology. Comparisons between biomedical genes such as EEF2 (elongation factor 2 human, mouse, etc), WDR85 in epigenetics, HAR1 in human specificity, DLG1 in cognitive skill, and HLA-C in mosquito bite immunology with EF Tu DNA sequences have accounted for the reported circular dichroism thermo-stability data systematically; the results also infer a relatively less volatility geologic time period from 2 to 3 Gyr from adaptation viewpoint. Comparison to Thermotoga maritima MSB8 and Psychrobacter shows that Thermus thermophilus HB8 EF-Tu calibration sequence could be an outlier, consistent with free energy calculation by NUPACK. Diagram methodology allows computer simulation studies and HAR1 shows about 0.5% probability from chimp to human in terms of diagram location, and SNP simulation results such as amoebic meningoencephalitis NAF1 suggest correlation. Extensions to the studies of the translation and transcription elongation factor sequences in Megavirus Chiliensis, Megavirus Lba and Pandoravirus show that the studied Pandoravirus sequence could be an outlier with the highest fractal dimension and lowest entropy, as compared to chicken as a deviant in the DNMT3A DNA methylation gene sequences from zebrafish to human and to the less than one percent probability in computer simulation using the HAR1 0.5% probability as reference. The diagram methodology would be useful in ancestral gene

  5. Elongation factor-1 alpha is a selective regulator of growth factor withdrawal and ER stress-induced apoptosis.

    PubMed

    Talapatra, S; Wagner, J D O; Thompson, C B

    2002-08-01

    To identify genes that contribute to apoptotic resistance, IL-3 dependent hematopoietic cells were transfected with a cDNA expression library and subjected to growth factor withdrawal. Transfected cells were enriched for survivors over two successive rounds of IL-3 withdrawal and reconstitution, resulting in the identification of a full-length elongation factor 1 alpha (EF-1alpha) cDNA. Ectopic EF-1alpha expression conferred protection from growth factor withdrawal and agents that induce endoplasmic reticulum stress, but not from nuclear damage or death receptor signaling. Overexpression of EF-1alpha did not lead to growth factor independent cell proliferation or global alterations in protein levels or rates of synthesis. These findings suggest that overexpression of EF-1alpha results in selective resistance to apoptosis induced by growth factor withdrawal and ER stress. PMID:12107828

  6. Gdown1 Associates Efficiently with RNA Polymerase II after Promoter Clearance and Displaces TFIIF during Transcript Elongation

    PubMed Central

    DeLaney, Elizabeth

    2016-01-01

    Pausing during the earliest stage of transcript elongation by RNA polymerase II (Pol II) is a nearly universal control point in metazoan gene expression. The substoichiometric Pol II subunit Gdown1 facilitates promoter proximal pausing in vitro in extract-based transcription reactions, out-competes the initiation/elongation factor TFIIF for binding to free Pol II and co-localizes with paused Pol II in vivo. However, we have shown that Gdown1 cannot functionally associate with the Pol II preinitiation complex (PIC), which contains TFIIF. In the present study, we determined at what point after initiation Gdown1 can associate with Pol II and how rapidly this competition with TFIIF occurs. We show that, as with the PIC, Gdown1 cannot functionally load into open complexes or complexes engaged in abortive synthesis of very short RNAs. Gdown1 can load into early elongation complexes (EECs) with 5–9 nt RNAs, but efficient association with EECs does not take place until the point at which the upstream segment of the long initial transcription bubble reanneals. Tests of EECs assembled on a series of promoter variants confirm that this bubble collapse transition, and not transcript length, modulates Gdown1 functional affinity. Gdown1 displaces TFIIF effectively from all complexes downstream of the collapse transition, but this displacement is surprisingly slow: complete loss of TFIIF stimulation of elongation requires 5 min of incubation with Gdown1. The relatively slow functional loading of Gdown1 in the presence of TFIIF suggests that Gdown1 works in promoter-proximal pausing by locking in the paused state after elongation is already antagonized by other factors, including DSIF, NELF and possibly the first downstream nucleosome. PMID:27716820

  7. Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

    PubMed Central

    Habben, J E; Moro, G L; Hunter, B G; Hamaker, B R; Larkins, B A

    1995-01-01

    Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals. Images Fig. 1 Fig. 2 PMID:7567989

  8. Phylogeny of the Glomerales and Diversisporales (fungi: Glomeromycota) from actin and elongation factor 1-alpha sequences.

    PubMed

    Helgason, Thorunn; Watson, Irene J; Young, J Peter W

    2003-12-01

    The arbuscular mycorrhizal (AM) fungi have been elevated to the phylum Glomeromycota based on a ribosomal gene phylogeny. In order to test this phylogeny, we amplified and sequenced small subunit ribosomal RNA (SSUrRNA), actin and elongation factor 1 (EF1)-alpha gene fragments from single spores of Acaulospora laevis, Glomus caledonium, Gigaspora margarita, and Scutellospora dipurpurescens. Sequence variation within and among spores of an isolate was low except for SSUrRNA in S. dipurpurescens, and the actin amino acid sequence was more conserved than that of EF1-alpha. The AM fungal sequences were more similar to one another than to any other fungal group. Joint phylogenetic analysis of the actin and EF1-alpha sequences suggested that the sister group to the AM fungi was a Zygomycete order, the Mortierellales.

  9. Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes.

    PubMed

    Kamaishi, T; Hashimoto, T; Nakamura, Y; Nakamura, F; Murata, S; Okada, N; Okamoto, K; Shimizu, M; Hasegawa, M

    1996-02-01

    Partial regions of the mRNA encoding a major part of translation elongation factor 1 alpha (EF-1 alpha) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1 alpha's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed, G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. PMID:8919877

  10. Elongation as a factor in artefacts of humans and other animals: an Acheulean example in comparative context

    PubMed Central

    Gowlett, J. A. J.

    2013-01-01

    Elongation is a commonly found feature in artefacts made and used by humans and other animals and can be analysed in comparative study. Whether made for use in hand or beak, the artefacts have some common properties of length, breadth, thickness and balance point, and elongation can be studied as a factor relating to construction or use of a long axis. In human artefacts, elongation can be traced through the archaeological record, for example in stone blades of the Upper Palaeolithic (traditionally regarded as more sophisticated than earlier artefacts), and in earlier blades of the Middle Palaeolithic. It is now recognized that elongation extends to earlier Palaeolithic artefacts, being found in the repertoire of both Neanderthals and more archaic humans. Artefacts used by non-human animals, including chimpanzees, capuchin monkeys and New Caledonian crows show selection for diameter and length, and consistent interventions of modification. Both chimpanzees and capuchins trim side branches from stems, and appropriate lengths of stave are selected or cut. In human artefacts, occasional organic finds show elongation back to about 0.5 million years. A record of elongation achieved in stone tools survives to at least 1.75 Ma (million years ago) in the Acheulean tradition. Throughout this tradition, some Acheulean handaxes are highly elongated, usually found with others that are less elongated. Finds from the million-year-old site of Kilombe and Kenya are given as an example. These findings argue that the elongation need not be integral to a design, but that artefacts may be the outcome of adjustments to individual variables. Such individual adjustments are seen in animal artefacts. In the case of a handaxe, the maker must balance the adjustments to achieve a satisfactory outcome in the artefact as a whole. It is argued that the need to make decisions about individual variables within multivariate objects provides an essential continuity across artefacts made by

  11. Dynamics of Elongation Factor 2 Kinase Regulation in Cortical Neurons in Response to Synaptic Activity

    PubMed Central

    Kenney, Justin W.; Sorokina, Oksana; Genheden, Maja; Sorokin, Anatoly

    2015-01-01

    The rapid regulation of cell signaling in response to calcium in neurons is essential for real-time processing of large amounts of information in the brain. A vital regulatory component, and one of the most energy-intensive biochemical processes in cells, is the elongation phase of mRNA translation, which is controlled by the Ca2+/CaM-dependent elongation factor 2 kinase (eEF2K). However, little is known about the dynamics of eEF2K regulation in neurons despite its established role in learning and synaptic plasticity. To explore eEF2K dynamics in depth, we stimulated synaptic activity in mouse primary cortical neurons. We find that synaptic activity results in a rapid, but transient, increase in eEF2K activity that is regulated by a combination of AMPA and NMDA-type glutamate receptors and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) pathways. We then used computational modeling to test the hypothesis that considering Ca2+-coordinated MEK/ERK, mTORC1, and eEF2k activation is sufficient to describe the observed eEF2K dynamics. Although such a model could partially fit the empirical findings, it also suggested that a crucial positive regulator of eEF2K was also necessary. Through additional modeling and empirical evidence, we demonstrate that AMP kinase (AMPK) is also an important regulator of synaptic activity-driven eEF2K dynamics in neurons. Our combined modeling and experimental findings provide the first evidence that it is necessary to consider the combined interactions of Ca2+ with MEK/ERK, mTORC1, and AMPK to adequately explain eEF2K regulation in neurons. PMID:25698741

  12. Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy

    PubMed Central

    Moore, Claire E.J.; Wang, Xuemin; Xie, Jianling; Pickford, Jo; Barron, John; Regufe da Mota, Sergio; Versele, Matthias; Proud, Christopher G.

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy. PMID:26795954

  13. Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

    PubMed Central

    Wang, Xuemin; Regufe da Mota, Sergio; Liu, Rui; Moore, Claire E.; Xie, Jianling; Lanucara, Francesco; Agarwala, Usha; Pyr dit Ruys, Sébastien; Vertommen, Didier; Rider, Mark H.; Eyers, Claire E.

    2014-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways. PMID:25182533

  14. Elongation factor 4 remodels the A-site tRNA on the ribosome

    PubMed Central

    Gagnon, Matthieu G.; Lin, Jinzhong; Steitz, Thomas A.

    2016-01-01

    During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant. PMID:27092003

  15. Direct evidence of an elongation factor-Tu/Ts·GTP·Aminoacyl-tRNA quaternary complex.

    PubMed

    Burnett, Benjamin J; Altman, Roger B; Ferguson, Angelica; Wasserman, Michael R; Zhou, Zhou; Blanchard, Scott C

    2014-08-22

    During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.

  16. Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

    PubMed

    Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

    2016-01-01

    Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

  17. SHORT HYPOCOTYL 1 encodes a SMARCA3-like chromatin remodeling factor regulating elongation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the mechanisms and control of hypocotyl elongation is important for greenhouse vegetable crop production. In this study, we identified SHORT HYPOCOTYL1 (SH1) in cucumber which regulates low-dosage ultraviolet B (LDUVB)-dependent hypocotyl elongation by recruiting the cucumber UVR8 sign...

  18. Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation.

    PubMed

    Gergics, Peter; Christian, Helen C; Choo, Monica S; Ajmal, Adnan; Camper, Sally A

    2016-09-01

    Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks. PMID:27580811

  19. Blastocystis elongation factor-1alpha: genomic organization, taxonomy and phylogenetic relationships.

    PubMed

    Ho, L C; Armiugam, A; Jeyaseelan, K; Yap, E H; Singh, M

    2000-08-01

    The elongation factor-1 alpha (EF-1alpha) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEalpha) that codes for a major part of the coding region of the EF-lalpha protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5' and 3' ends of the EF-1alpha gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5' end of B. hominis C EF-1alpha were identified. A comparative study on the deduced amino acid sequences of BEalpha of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the species. A phylogenetic reconstruction analysis with other eukaryotic EF-1alpha sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms. PMID:11085233

  20. Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B*

    PubMed Central

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-01-01

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. PMID:25635048

  1. Elongation Factor P and Modifying Enzyme PoxA Are Necessary for Virulence of Shigella flexneri

    PubMed Central

    Marman, Hannah E.; Mey, Alexandra R.

    2014-01-01

    Elongation factor P (EF-P) is a universally conserved bacterial translation factor. In many bacteria, EF-P is posttranslationally modified by PoxA, which covalently attaches a β-lysine to a conserved lysine residue of EF-P. Here we show that both EF-P and PoxA are necessary for virulence of the human diarrheal pathogen Shigella flexneri. Loss of either EF-P or PoxA leads to an impaired ability of S. flexneri to invade epithelial cells and form plaques in an epithelial cell monolayer. Proteomic analysis of efp and poxA deletion mutants revealed decreased levels of several virulence effector proteins, including IpaA, -B, and -C and IcsA. Additionally, mRNA levels of virB and virF, which encode master virulence regulators, were decreased in the efp mutant. The reduction in virF transcription was at least partially due to decreased levels of CpxA, which activates virF through the response regulator CpxR. The role of CpxAR in reduced synthesis of VirF and its downstream effectors was indicated by restoration of invasion when a mutation resulting in constitutively activated CpxR was introduced into the efp mutant. Thus, modified EF-P is required for appropriate synthesis of proteins involved in the virulence of this bacterial pathogen. PMID:24935977

  2. The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

    PubMed Central

    Goldman, Seth R; Nair, Nikhil U; Wells, Christopher D; Nickels, Bryce E; Hochschild, Ann

    2015-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001 PMID:26371553

  3. Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase.

    PubMed

    Will, Nathan; Piserchio, Andrea; Snyder, Isaac; Ferguson, Scarlet B; Giles, David H; Dalby, Kevin N; Ghose, Ranajeet

    2016-09-27

    Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, which is predicted to contain several SEL-1-like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution nuclear magnetic resonance methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (αI) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (αII-αIV). The helical core is structurally similar to members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, αII and αIII, comprise the TPR, and the last helix, αIV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing αIV and, therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding nor isolated eEF-2K627-725 inhibits eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.

  4. Evolution of elongation factor-like (EFL) protein in Rhizaria is revised by radiolarian EFL gene sequences.

    PubMed

    Ishitani, Yoshiyuki; Kamikawa, Ryoma; Yabuki, Akinori; Tsuchiya, Masashi; Inagaki, Yuji; Takishita, Kiyotaka

    2012-01-01

    Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study.

  5. Evolution of elongation factor-like (EFL) protein in Rhizaria is revised by radiolarian EFL gene sequences.

    PubMed

    Ishitani, Yoshiyuki; Kamikawa, Ryoma; Yabuki, Akinori; Tsuchiya, Masashi; Inagaki, Yuji; Takishita, Kiyotaka

    2012-01-01

    Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study. PMID:22672006

  6. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

    PubMed

    Scotti, John S; Leung, Ivanhoe K H; Ge, Wei; Bentley, Michael A; Paps, Jordi; Kramer, Holger B; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M; Bowman, Lesley A H; Loik, Nikita D; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J; Tang, Christoph M; Claridge, Timothy D W; Preston, Gail M; McDonough, Michael A; Schofield, Christopher J

    2014-09-16

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

  7. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

    PubMed

    Scotti, John S; Leung, Ivanhoe K H; Ge, Wei; Bentley, Michael A; Paps, Jordi; Kramer, Holger B; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M; Bowman, Lesley A H; Loik, Nikita D; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J; Tang, Christoph M; Claridge, Timothy D W; Preston, Gail M; McDonough, Michael A; Schofield, Christopher J

    2014-09-16

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins. PMID:25197067

  8. Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin.

    PubMed

    Lee, Kwangwoon; Alphonse, Sébastien; Piserchio, Andrea; Tavares, Clint D J; Giles, David H; Wellmann, Rebecca M; Dalby, Kevin N; Ghose, Ranajeet

    2016-09-01

    Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells. PMID:27499441

  9. Trypanosoma cruzi elongation factor 1-alpha: nuclear localization in parasites undergoing apoptosis.

    PubMed

    Billaut-Mulot, O; Fernandez-Gomez, R; Loyens, M; Ouaissi, A

    1996-09-26

    The cloning and sequencing of the gene coding for Trypanosoma cruzi elongation factor 1 alpha (TcEF-1 alpha) was performed by screening a T. cruzi genomic library with a probe obtained through the polymerase chain reaction (PCR) amplification of T. cruzi DNA using two oligonucleotides deduced from the sequence of T. brucei EF-1 alpha. Southern blot analysis of T. cruzi digested genomic DNA and Northern blot hybridized with the labeled probe revealed that one copy of TcEF-1 alpha exist in the genome of the parasite. Indirect immunofluorescence technique using anti-EF-1 alpha antibodies and epimastigotes harvested after different days of in vitro culture showed that EF-1 alpha is localised in the cytoplasm of the parasites from the exponential growth phase. Surprisingly, during the stationary phase (ageing parasites), EF-1 alpha was found in the nucleus. Furthermore, treatment of parasites with the antibiotic drug geneticin (G418) which induces the death of epimastigotes by apoptosis showed selective localization of EF-1 alpha in the nucleus of dying parasites. This observation supports the notion already reported in the case of mammalian cells that EF-1 alpha could participate in the transcription processes and possibly in the case of T. cruzi, in the expression regulation of genes involved in the control of cell death. The possible transfection and genomic manipulation of T. cruzi may provide a model to study the role of TcEF-1 alpha in this phenomenon. PMID:8863724

  10. Expression of elongation factor-1 alpha and S1 in young and old human skeletal muscle.

    PubMed

    Welle, S; Thornton, C; Bhatt, K; Krym, M

    1997-09-01

    Previous research has indicated that reduced expression of elongation factor-1 alpha (EF-1 alpha) may be an important determinant of the reduced rate of protein synthesis in senescent animals and cultured cells. The present study examined whether expression of EF-1 alpha or S1, a homologous protein found exclusively in postmitotic tissues, is reduced in senescent human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis muscles of healthy young (22-31 yr old) and old (61-74 yr old) subjects. As reported previously, myofibrillar protein synthesis was approximately 40% slower in the older muscle (p < .001) as determined by incorporation of a stable isotope. Immunoblotting revealed no difference in the concentration of EF-1 alpha + S1 between younger and older muscle. RT-PCR assays indicated that S1 mRNA was much more abundant than EF-1 alpha mRNA in muscles of both age groups, with no reduction in either EF-1 alpha or S1 mRNA abundance in older muscles. We conclude that expression of EF-1 alpha and S1 is not diminished in older muscles and does not explain the age-related slowing of protein synthesis in human skeletal muscle. However, we cannot exclude the possibility that the activity of these proteins declines during senescence due to post-translational modifications. PMID:9310071

  11. Cloning and expression of Bombyx mori silk gland elongation factor 1gamma in Escherichia coli.

    PubMed

    Kamiie, Katsuyoshi; Nomura, Yoshitaka; Kobayashi, Satoru; Taira, Hideharu; Kobayashi, Kohmei; Yamashita, Tetsuro; Kidou, Shin-ichiro; Ejiri, Shin-ichiro

    2002-03-01

    Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of alpha-, beta-, gamma-, and delta-subunits. EF-1alpha GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP. EF-1betagammadelta catalyzes the exchange of EF-1alpha-bound GDP for exogenous GTP and stimulates the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosomes. EF-1gamma cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library. The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively. The silk gland EF-1gamma shares 67.3% amino acid identity with Artemia salina EF-lgamma. The N-terminal domain (amino acid residues 1-211) of silk gland EF-lgamma is 29.3% identical to maize glutathione S-transferase. We demonstrated that silk gland EF-lgamma bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1gamma may have the capacity to bind to glutathione. PMID:12005049

  12. Divergence among Genes Encoding the Elongation Factor Tu of Yersinia Species▿

    PubMed Central

    Isabel, Sandra; Leblanc, Éric; Boissinot, Maurice; Boudreau, Dominique K.; Grondin, Myrian; Picard, François J.; Martel, Eric A.; Parham, Nicholas J.; Chain, Patrick S. G.; Bader, Douglas E.; Mulvey, Michael R.; Bryden, Louis; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    2008-01-01

    Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species. PMID:18790860

  13. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  14. Affinity labeling of eukaryotic elongation factors using N epsilon-bromoacetyl-Lys-tRNA.

    PubMed Central

    Johnson, A E; Slobin, L I

    1980-01-01

    eEF-T and eEF-Tu from rabbit reticulocyte and from Artemia were affinity labeled using N epsilon-bromoacetyl-Lys-tRNA prepared with either yeast or E. coli tRNA. Only the eEF-Tu polypeptide was crosslinked when eEF-T was incubated with the reactive aminoacyl-tRNA analogue, which indicates that at least part of the aminoacyl-tRNA binding site is the same in both eEF-Tu and the multisubunit eEF-T. Complex formation (eEF-Tu x aa-tRNA x GTP) was required for crosslinking, since no covalent reaction with eEF-Tu occurred in the absence of GTP. The yield of crosslinked product was greatly reduced by adding either unmodified rabbit liver aminoacyl-tRNA or unmodified E. coli Lys-tRNA to the incubation to compete for the aminoacyl-tRNA binding site on eEF-T or eEF-Tu, indicating that the covalent reaction occurs while the N epsilon-bromoacetyl-Lys-tRNA is bound in this site. The affinity labeling of a prokaryotic and two different eukaryotic elongation factors by the same reagent suggests that there may be conservation of structure in the region of the proteins which binds the aminoacyl end of the aminoacyl-tRNA. PMID:7001363

  15. Three genes for the elongation factor EF-1 alpha in Mucor racemosus.

    PubMed

    Linz, J E; Katayama, C; Sypherd, P S

    1986-02-01

    We cloned three genes from Mucor racemosus coding for protein synthesis elongation factor 1 alpha (EF-1 alpha). A 110-base-pair (bp) EF-1 alpha-specific cDNA clone was identified by hybrid-selected translation. The nucleotide sequence of the cDNA showed significant homology to a region of the Saccharomyces cerevisiae genes for EF-1 alpha (TEF1 and TEF2). The cDNA was used to isolate an 850-bp EcoRI genomic DNA fragment containing a portion of the EF-1 alpha gene. Screening of a lambda/M. racemosus genomic DNA bank with the 850-bp EcoRI probe resulted in the identification of three DNA fragments containing a common 850-bp EcoRI fragment within a short overlapping region. S1 nuclease analysis of the three EF-1 alpha DNA fragments showed that the EF-1 alpha transcript covered the short overlapping region in the clones. Restriction fragments purified from flanking regions in each clone were used to probe a HindIII digest of M. racemosus genomic DNA. Each flanking probe hybridized to one of three DNA fragments which hybridized to the 850-bp EF-1 alpha-specific probe. Nucleotide sequence data from two random "shotgun clones" of one of the three genes show good homology to two regions of S. cerevisiae TEF1. The data indicate the presence of three genes for EF-1 alpha in M. racemosus located at unique sites in the genome. PMID:2946933

  16. Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene.

    PubMed

    Hasegawa, Eri; Ota, Yuko; Hattori, Tsutomu; Kikuchi, Taisei

    2010-01-01

    We analyzed the sequences of three DNA regions-the translation elongation factor-1 alpha (EF-1 alpha) gene and the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA-to compare their accuracy in identifying species of Japanese Armillaria. We studied 49 isolates of eight Armillaria species, A. mellea, A. ostoyae, A. nabsnona, A. cepistipes, A. gallica, A. sinapina, A. tabescens and the biological species Nagasawa E (Nag. E). Phylogenetic analyses of the ITS and IGS data helped in identifying A. mellea, A. ostoyae, A. nabsnona, A. tabescens and Nag. E but could not be used to identify A. gallica, A. cepistipes and A. sinapina. Nevertheless our analysis showed that the EF-1 alpha gene was clearly different in the eight examined species. Restriction fragment length polymorphisms (RFLP) of the IGS-1 region could be used to distinguish most species, but the RFLP profiles of some isolates of A. cepistipes and A. sinapina were the same even with four different restriction enzymes. In conclusion, among the techniques examined in this study, analyzing the EF-1 alpha sequence was found to be the most suitable method for identifying different species of Japanese Armillaria. PMID:20648756

  17. Recombination between elongation factor 1α genes from distantly related archaeal lineages

    PubMed Central

    Inagaki, Yuji; Susko, Edward; Roger, Andrew J.

    2006-01-01

    Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1α (EF-1α) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1α gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1α gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential “informational” genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing. PMID:16537397

  18. Eukaryotic Translation Elongation Factor 1A Induces Anoikis by Triggering Cell Detachment*

    PubMed Central

    Itagaki, Keisuke; Naito, Toshihiko; Iwakiri, Ryota; Haga, Makoto; Miura, Shougo; Saito, Yohei; Owaki, Toshiyuki; Kamiya, Sadahiro; Iyoda, Takuya; Yajima, Hirofumi; Iwashita, Shintaro; Ejiri, Shin-Ichiro; Fukai, Fumio

    2012-01-01

    Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing β1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage. PMID:22399298

  19. Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

    PubMed Central

    Koenigs, Arno; Zipfel, Peter F.; Kraiczy, Peter

    2015-01-01

    Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system. PMID:26230848

  20. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

  1. Do maise and wheat chloroplast protein synthesis elongation factor, EF-Tu, protect Rubisco activase from thermal aggregation and inactivation?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize (Zea mays L.) chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in the development of heat tolerance. The precursor of this protein (pre-EF-Tu) has been shown to display chaperone activity, as it protected heat labile citrate synthase and malate dehydrogenase from the...

  2. Heat tolerance and expression of protein synthesis elongation factors, EF-Tu and EF-1a, in spring wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein elongation factors, EF-Tu and EF-1a, have been implicated in cell response to heat stress. In spring wheat, EF-Tu displays chaperone activity and reduces thermal aggregation of Rubisco activase. Similarly, in mammalian cells, EF-1a displays chaperone-like activity and regulates the expressio...

  3. Role of Testicular Luminal Factors on Basal Cell Elongation and Proliferation in the Mouse Epididymis1

    PubMed Central

    Kim, Bongki; Roy, Jeremy; Shum, Winnie W.C.; Da Silva, Nicolas; Breton, Sylvie

    2014-01-01

    ABSTRACT A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens. PMID:25411392

  4. Rooting for the root of elongation factor-like protein phylogeny.

    PubMed

    Kamikawa, Ryoma; Sakaguchi, Miako; Matsumoto, Takuya; Hashimoto, Tetsuo; Inagaki, Yuji

    2010-09-01

    Lateral gene transfer (LGT) may play a pivotal role in the evolution of elongation factor-like (EFL) genes in eukaryotes. To date, numbers of putative cases for lateral transfer of EFL genes have been postulated based on unrooted EFL phylogenies. Nevertheless, the root position in EFL phylogeny is important to validate lateral EFL gene transfer: for instance, a clade of two EFL homologs from distantly related organisms in an unrooted EFL tree does not necessarily confirm the LGT, since the possibility that the root may locate in this clade cannot be excluded. Cocquyt et al. (2009, p. 39) recently demonstrated that a putative case of lateral EFL gene transfer, which was originally proposed based on an unrooted phylogeny, could not be endorsed by the corresponding rooted analysis. Although rooting EFL phylogeny is indispensable to elucidate various aspects in EFL gene evolution, we suspected that the outgroup clade comprised of EF-1alpha and eukaryote-specific EF-1alpha paralogs erroneously attached to long EFL branches in Cocquyt et al. (2009) - a typical long branch attraction (LBA) artifact. Here, we systematically assessed the putative LBA artifact between the branch leading to the outgroup clade and long ingroup branches by analyzing the original dataset used in Cocquyt et al. (2009) with and without modifying ingroup-sequence sampling. A series of the rooted EFL analyses indicated that the root inference was highly susceptible to presence and absence of long-branched ingroup-sequences, suggesting that the rooted EFL phylogenies cannot be free from severe LBA artifact. We also discussed a new aspect in EFL gene evolution in stramenopiles identified in the course of the EFL analyses described above. Finally, the relative timing of the first emergence of EFL gene in eukaryotes was contemplated based on the current EF-1alpha/EFL distribution.

  5. Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors.

    PubMed

    Warren, Kylie; Wei, Ting; Li, Dongsheng; Qin, Fangyun; Warrilow, David; Lin, Min-Hsuan; Sivakumaran, Haran; Apolloni, Ann; Abbott, Catherine M; Jones, Alun; Anderson, Jenny L; Harrich, David

    2012-06-12

    Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.

  6. Cloning and Sequencing of the cDNA Encoding the Rubber Elongation Factor of Hevea brasiliensis

    PubMed Central

    Goyvaerts, Elisabeth; Dennis, Mark; Light, David; Chua, Nam-Hai

    1991-01-01

    In Hevea brasiliensis, the rubber particle in the laticiferous vessel is the site of rubber (cis-1-4-polyisoprene) biosynthesis. A 14 kilodalton protein, rubber elongation factor (REF), is associated with the rubber particle in a ratio of one REF to one rubber molecule (Dennis M, Henzel W, Bell J, Kohr W, Light D [1989] J Biol Chem 264: 18618-18628; Dennis M, Light D [1989] J Biol Chem 264: 18608-18617). To obtain more information concerning the function of REF and its synthesis and assembly in the rubber particle, we isolated cDNA clones encoding REF. We used antibodies to REF to screen a Hevea leaf γgt11 cDNA expression library and obtained several positive clones. Sequence analysis of the REF cDNA clones showed that the REF mRNA contains 121 nucleotides of 5′-nontranslated sequences and a 205 nucleotide 3′-nontranslated region. The open reading frame encodes the entire 14 kilodalton REF protein without any extra amino acids (Dennis M, Henzel W, Bell J, Kohr W, Light D [1989] J Biol Chem 264: 18618-18628). The REF cDNA was subcloned in pGEM-3Z/-4Z and expressed in vitro. The translation product is a 14 kilodalton protein that can be immunoprecipitated with antibodies to REF. Addition of microsomal membranes to the in vitro translation product did not alter the mobility of the REF protein. This, and the sequence data, indicate that REF is not made as a preprotein. Our results suggest that REF is synthesized on free polysomes in the laticifer cytoplasm and that assembly of the rubber particles is likely to occur in the cytosol. ImagesFigure 2Figure 3 PMID:16668388

  7. Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Sutherland, Brian G; Robson, Debra L; Arya, Rigya; Kelly, Karen; Jacobs, René L; Borradaile, Nica M

    2016-09-01

    Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease. PMID:27613825

  8. Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

    PubMed

    Cetin, R; Krab, I M; Anborgh, P H; Cool, R H; Watanabe, T; Sugiyama, T; Izaki, K; Parmeggiani, A

    1996-05-15

    This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.

  9. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-Ichi; Yokoyama, Shigeyuki

    2015-10-15

    Selenocysteine (Sec), the 21(st) amino acid in translation, uses its specific tRNA (tRNA(Sec)) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1-3), followed by four winged-helix domains (WHD1-4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA(Sec) is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA(Sec), SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA(Sec) allows SelB to specifically recognize tRNA(Sec) and characteristically place it at the ribosomal A-site. PMID:26304550

  10. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

    PubMed Central

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2015-01-01

    Selenocysteine (Sec), the 21st amino acid in translation, uses its specific tRNA (tRNASec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNASec (Sec-tRNASec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNASec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNASec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNASec allows SelB to specifically recognize tRNASec and characteristically place it at the ribosomal A-site. PMID:26304550

  11. Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Sutherland, Brian G; Robson, Debra L; Arya, Rigya; Kelly, Karen; Jacobs, René L; Borradaile, Nica M

    2016-09-01

    Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.

  12. Identification of Tetrahymena 14-nm filament-associated protein as elongation factor 1 alpha.

    PubMed

    Kurasawa, Y; Numata, O; Katoh, M; Hirano, H; Chiba, J; Watanabe, Y

    1992-11-01

    Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during conjugation. By immunoblotting using monoclonal and polyclonal antibodies following two-dimensional gel electrophoresis, we demonstrated that the 14-nm filament protein fraction contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0; a monoclonal antibody (MAb) 26B4 and a polyclonal antibody 49KI reacted only to a pI 8.0 protein, while two other MAbs, 11B6 and 11B8, reacted only to a pI 9.0 protein. From the N-terminal amino acid sequences, the pI 8.0 protein was identified as the previously reported 14-nm filament-forming protein/citrate synthase, but the pI 9.0 protein N-terminal sequence had no similarity with that of the pI 8.0 protein. The pI 9.0 protein is considered to be a 14-nm filament-associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a Tetrahymena pyriformis cDNA library, we identified the pI 9.0 protein as elongation factor 1 alpha (EF-1 alpha) based on it sharing 73-76% sequence identity with EF-1 alpha from several species. PMID:1385189

  13. Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa

    PubMed Central

    Rajkovic, Andrei; Erickson, Sarah; Witzky, Anne; Branson, Owen E.; Seo, Jin; Gafken, Philip R.; Frietas, Michael A.; Whitelegge, Julian P.; Faull, Kym F.; Navarre, William; Darwin, Andrew J.

    2015-01-01

    ABSTRACT Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival. PMID:26060278

  14. Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II

    SciTech Connect

    Rappaport, J.; Cho, K.; Saltzman, A.; Prenger, J.; Golomb, M.; Weinmann, R.

    1988-08-01

    Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the ..beta..-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of ..beta..-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while ..beta..-galactosidase had no effect. The effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that anitibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

  15. Organization and nucleotide sequence of a gene cluster comprising the translation elongation factor 1 alpha, ribosomal protein S10 and tRNA(Ala) from Halobacterium halobium.

    PubMed

    Fujita, T; Itoh, T

    1995-09-01

    Lambda EMBL clone containing a gene cluster coding for the translation elongation factor 1alpha, ribosomal protein S10 and tRNA(ala) was identified in a genomic library for the halophilic archaebacterium Halobacterium halobium using a PCR probe amplified by two oligonucleotide primers for conserved amino acid sequences of the elongation factor 1 alpha family. The gene coding for elongation factor EF-2 was also found 4.3kb upstream from the 5'end of the elongation factor 1 alpha by hybridization analysis using a DNA fragment specific for EF-2 from Halobacterium halobium [1]. Halobacterial and eukaryotic elongation factor 1 alpha homologues are very similar in sequence and in length and appear to be more closely related to each other than to the eubacterial protein. PMID:8653072

  16. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect. PMID:26587907

  17. Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

    PubMed

    Brigotti, M; Rambelli, F; Zamboni, M; Montanaro, L; Sperti, S

    1989-02-01

    alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system. PMID:2930482

  18. The chvH locus of Agrobacterium encodes a homologue of an elongation factor involved in protein synthesis.

    PubMed

    Peng, W T; Banta, L M; Charles, T C; Nester, E W

    2001-01-01

    The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli. The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium. PMID:11114898

  19. Molecular characterization and expression analysis of elongation factors 1A and 2 from the Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Wang, Lei; Liu, Yuan; Wang, Wei-Na; Mai, Wei-Jun; Xin, Yu; Zhou, Jun; He, Wen-Yin; Wang, An-Li; Sun, Ru-Yong

    2011-03-01

    Elongation factors (EF) are abundant cell proteins that play important roles in the metabolism of all multicellular organisms. Here we describe a functional analysis of elongation factor 1-alpha (EF1A) and elongation factor 2 (EF2), from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNAs of genes corresponding to EF1A and EF2 were obtained that were 1547 and 2729 bp long, with open reading frames encoding 461 and 846 amino acids, respectively. The deduced amino acid sequences of L. vannamei EF1A and EF2 showed high similarity with those from mice, humans, chickens and other shrimps. RT-PCR analysis indicated that mRNA transcripts of EF1A and EF2 are strongly (but differentially) expressed in haemocytes and gill tissue, and at varying levels in other examined tissues, of the shrimps. Levels of both EF1A and EF2 transcripts increased when shrimps were challenged by pH and cadmium stress, but reached maximal levels after different exposure periods. These results indicate that EF1A and EF2 may play distinct, essential roles in the repair of cellular damage induced by pH and cadmium stress. PMID:20857205

  20. Thermoperiodic control of hypocotyl elongation depends on auxin-induced ethylene signaling that controls downstream PHYTOCHROME INTERACTING FACTOR3 activity.

    PubMed

    Bours, Ralph; Kohlen, Wouter; Bouwmeester, Harro J; van der Krol, Alexander

    2015-02-01

    We show that antiphase light-temperature cycles (negative day-night temperature difference [-DIF]) inhibit hypocotyl growth in Arabidopsis (Arabidopsis thaliana). This is caused by reduced cell elongation during the cold photoperiod. Cell elongation in the basal part of the hypocotyl under -DIF was restored by both 1-aminocyclopropane-1-carboxylic acid (ACC; ethylene precursor) and auxin, indicating limited auxin and ethylene signaling under -DIF. Both auxin biosynthesis and auxin signaling were reduced during -DIF. In addition, expression of several ACC Synthase was reduced under -DIF but could be restored by auxin application. In contrast, the reduced hypocotyl elongation of ethylene biosynthesis and signaling mutants could not be complemented by auxin, indicating that auxin functions upstream of ethylene. The PHYTOCHROME INTERACTING FACTORS (PIFs) PIF3, PIF4, and PIF5 were previously shown to be important regulators of hypocotyl elongation. We now show that, in contrast to pif4 and pif5 mutants, the reduced hypocotyl length in pif3 cannot be rescued by either ACC or auxin. In line with this, treatment with ethylene or auxin inhibitors reduced hypocotyl elongation in PIF4 overexpressor (PIF4ox) and PIF5ox but not PIF3ox plants. PIF3 promoter activity was strongly reduced under -DIF but could be restored by auxin application in an ACC Synthase-dependent manner. Combined, these results show that PIF3 regulates hypocotyl length downstream, whereas PIF4 and PIF5 regulate hypocotyl length upstream of an auxin and ethylene cascade. We show that, under -DIF, lower auxin biosynthesis activity limits the signaling in this pathway, resulting in low activity of PIF3 and short hypocotyls.

  1. Lignification in rapidly elongating internodes of deep water rice as a limiting factor in growth

    SciTech Connect

    Sauter, M.; Kende, H. )

    1990-05-01

    Internodes of deep water rice are induced to elongate rapidly by partial submergence, or by treatment with ethylene or gibberellin. This growth response is based, in part, on enhanced cell elongation and an increase in the size of the internodal growing zone. For this to occur, processes that limit growth, e.g. lignification, must be delayed. We examined the activity and distribution of two enzymes of the lignin biosynthetic pathway, phenylalanine ammonia-lyase (PAL) and coniferylalcohol dehydrogenase (CAD) in rapidly growing and control internodes. CAD activity decreased in the rapidly growing region of submerged or gibberellin-treated internodes to about 25% of the activity found in air-grown control internodes. No comparable change in CAD activity was observed in the older, non-growing portions of the internodes. PAL activity changed in similar fashion upon induction of rapid growth.

  2. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

    PubMed Central

    Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

    2014-01-01

    Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

  3. GbTCP, a cotton TCP transcription factor, confers fibre elongation and root hair development by a complex regulating system.

    PubMed

    Hao, Juan; Tu, Lili; Hu, Haiyan; Tan, Jiafu; Deng, Fenglin; Tang, Wenxin; Nie, Yichun; Zhang, Xianlong

    2012-10-01

    As the most important natural raw material for textile industry, cotton fibres are an excellent model for studying single-cell development. Although expression profiling and functional genomics have provided some data, the mechanism of fibre development is still not well known. A class I TCP transcription factor (designated GbTCP), encoding 344 amino acids, was isolated from the normalized cDNA library of sea-island cotton fibre (from -2 to 25 days post anthesis). GbTCP was preferentially expressed in the elongating cotton fibre from 5 to 15 days post anthesis. Some expression was also observed in stems, apical buds, and petals. RNAi silencing of GbTCP produced shorter fibre, a reduced lint percentage, and a lower fibre quality than the wild-type plants. Overexpression of GbTCP enhanced root hair initiation and elongation in Arabidopsis and regulated branching. Solexa sequencing and Affymetrix GeneChip analysis indicated that GbTCP positively regulates the level of jasmonic acid (JA) and, as a result, activates downstream genes (reactive oxygen species, calcium signalling, ethylene biosynthesis and response, and several NAC and WRKY transcription factors) necessary for elongation of fibres and root hairs. JA content analysis in cotton also confirmed that GbTCP has a profound effect on JA biosynthesis. In vitro ovule culture showed that an appropriate concentration of JA promoted fibre elongation. The results suggest that GbTCP is an important transcription factor for fibre and root hair development by regulating JA biosynthesis and response and other pathways, including reactive oxygen species, calcium channel and ethylene signalling.

  4. Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

    PubMed

    Walker, John; Acestor, Nathalie; Gongora, Rafael; Quadroni, Manfredo; Segura, Iris; Fasel, Nicolas; Saravia, Nancy G

    2006-02-01

    Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL. PMID:16325936

  5. Mast Cell-Derived Tumor Necrosis Factor Can Promote Nerve Fiber Elongation in the Skin during Contact Hypersensitivity in Mice

    PubMed Central

    Kakurai, Maki; Monteforte, Rossella; Suto, Hajime; Tsai, Mindy; Nakae, Susumu; Galli, Stephen J.

    2006-01-01

    In humans, lesions of contact eczema or atopic dermatitis can exhibit increases in epidermal nerves, but the mechanism resulting in such nerve elongation are not fully understood. We found that contact hypersensitivity reactions to oxazolone in mice were associated with significant increases in the length of nerves in the epidermis and dermis. Using genetically mast cell-deficient c-kit mutant mice selectively repaired of their dermal mast cell deficiency with either wild-type or tumor necrosis factor (TNF)-deficient mast cells, we found that mast cells, and mast cell-derived TNF, significantly contributed to the elongation of epidermal and dermal PGP 9.5+ nerves and dermal CGRP+ nerves, as well as to the inflammation observed at sites of contact hypersensitivity in response to oxazolone. Moreover, the percentage of mast cells in close proximity to dermal PGP 9.5+ nerve fibers was significantly higher in wild-type mice and in c-kit mutant mice repaired of their dermal mast cell deficiency by the adoptive transfer of wild-type mast cells than in TNF-deficient mice or in TNF−/− mast cell-engrafted c-kit mutant mice. These observations show that mast cells, and mast cell-derived TNF, can promote the elongation of cutaneous nerve fibers during contact hypersensitivity in the mouse. PMID:17071594

  6. Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases.

    PubMed

    Liu, Rui; Proud, Christopher G

    2016-03-01

    Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual protein kinase that regulates the elongation stage of protein synthesis by phosphorylating and inhibiting its only known substrate, eEF2. Elongation is a highly energy-consuming process, and eEF2K activity is tightly regulated by several signaling pathways. Regulating translation elongation can modulate the cellular energy demand and may also control the expression of specific proteins. Growing evidence links eEF2K to a range of human diseases, including cardiovascular conditions (atherosclerosis, via macrophage survival) and pulmonary arterial hypertension, as well as solid tumors, where eEF2K appears to play contrasting roles depending on tumor type and stage. eEF2K is also involved in neurological disorders and may be a valuable target in treating depression and certain neurodegenerative diseases. Because eEF2K is not required for mammalian development or cell viability, inhibiting its function may not elicit serious side effects, while the fact that it is an atypical kinase and quite distinct from the vast majority of other mammalian kinases suggests the possibility to develop it into compounds that inhibit eEF2K without affecting other important protein kinases. Further research is needed to explore these possibilities and there is an urgent need to identify and characterize potent and specific small-molecule inhibitors of eEF2K. In this article we review the recent evidence concerning the role of eEF2K in human diseases as well as the progress in developing small-molecule inhibitors of this enzyme. PMID:26806303

  7. Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases

    PubMed Central

    Liu, Rui; Proud, Christopher G

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual protein kinase that regulates the elongation stage of protein synthesis by phosphorylating and inhibiting its only known substrate, eEF2. Elongation is a highly energy-consuming process, and eEF2K activity is tightly regulated by several signaling pathways. Regulating translation elongation can modulate the cellular energy demand and may also control the expression of specific proteins. Growing evidence links eEF2K to a range of human diseases, including cardiovascular conditions (atherosclerosis, via macrophage survival) and pulmonary arterial hypertension, as well as solid tumors, where eEF2K appears to play contrasting roles depending on tumor type and stage. eEF2K is also involved in neurological disorders and may be a valuable target in treating depression and certain neurodegenerative diseases. Because eEF2K is not required for mammalian development or cell viability, inhibiting its function may not elicit serious side effects, while the fact that it is an atypical kinase and quite distinct from the vast majority of other mammalian kinases suggests the possibility to develop it into compounds that inhibit eEF2K without affecting other important protein kinases. Further research is needed to explore these possibilities and there is an urgent need to identify and characterize potent and specific small-molecule inhibitors of eEF2K. In this article we review the recent evidence concerning the role of eEF2K in human diseases as well as the progress in developing small-molecule inhibitors of this enzyme. PMID:26806303

  8. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    PubMed

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  9. The Cotton Transcription Factor TCP14 Functions in Auxin-Mediated Epidermal Cell Differentiation and Elongation1[C][W

    PubMed Central

    Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

    2013-01-01

    Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells. PMID:23715527

  10. The distribution of Elongation Factor-1 Alpha (EF-1alpha), Elongation Factor-Like (EFL), and a non-canonical genetic code in the ulvophyceae: discrete genetic characters support a consistent phylogenetic framework.

    PubMed

    Gile, Gillian H; Novis, Philip M; Cragg, David S; Zuccarello, Giuseppe C; Keeling, Patrick J

    2009-01-01

    The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1alpha (EF-1alpha) while most Chlorophyta instead possess the related GTPase Elongation Factor-Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non-canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF-1alpha, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF-1alpha of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non-canonical genetic code previously described in A. acetabulum was observed in EF-1alpha sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis, and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.

  11. 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

  12. Sequences of elongation factors-1 alpha and -1 gamma and stimulation by juvenile hormone in Locusta migratoria.

    PubMed

    Zhou, S; Zhang, J; Fam, M D; Wyatt, G R; Walker, V K

    2002-11-01

    Two cDNAs encoding the alpha and gamma subunits of translation elongation factor-1 (EF-1) have been cloned and sequenced from the African migratory locust, Locusta migratoria. Southern blotting and real-time PCR analyses indicated that these sequences represent single copy genes. Comparison with sequences from other species indicated greater conservation for EF-1 alpha than for EF-1 gamma. The developmental profiles for EF-1 alpha and -1 gamma mRNA expression in the fat body paralleled reported changes in the hemolymph juvenile hormone (JH) titer in the fifth instar and were elevated during early reproductive maturation in the female adult. In maturing adults, there was a greater accumulation of EF-1 alpha and -1 gamma transcripts in females than in males. The levels of both transcripts were greatly increased by an enriched diet, previously shown to elevate JH titers and accelerate vitellogenin production. Treating JH-deprived adult females with the JH analog, methoprene, resulted in more than doubling of transcript levels of both genes, supporting the hypothesis that JH could stimulate the accumulation of LmEF-1 alpha and -1 gamma transcripts. We suggest that production of elongation factors, increased by JH, may contribute to the massive protein synthesis required for egg production. PMID:12530224

  13. Elongation factor SII-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein.

    PubMed Central

    Reines, D; Mote, J

    1993-01-01

    In eukaryotes the genetic material is contained within a coiled, protein-coated structure known as chromatin. RNA polymerases must recognize specific nucleoprotein assemblies and maintain contact with the underlying DNA duplex for many thousands of base pairs. Template-bound lac operon repressor from Escherichia coli arrests RNA polymerase II in vitro and in vivo [Kuhn, A., Bartsch, I. & Grummt, I. (1990) Nature (London) 344, 559-562; Deuschele, U., Hipskind, R. A. & Bujard, H. (1990) Science 248, 480-483]. We show that in a reconstituted transcription system, elongation factor SII enables RNA polymerase II to proceed through this blockage at high efficiency. lac repressor-arrested elongation complexes display an SII-activated transcript cleavage reaction, an activity associated with transcriptional read-through of a previously characterized region of bent DNA. This demonstrates factor-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. Nascent transcript cleavage may be a general mechanism by which RNA polymerase II can bypass many transcriptional impediments. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8446609

  14. Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex

    PubMed Central

    Carelli, Jordan D; Sethofer, Steven G; Smith, Geoffrey A; Miller, Howard R; Simard, Jillian L; Merrick, William C; Jain, Rishi K; Ross, Nathan T; Taunton, Jack

    2015-01-01

    Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products. DOI: http://dx.doi.org/10.7554/eLife.10222.001 PMID:26651998

  15. Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.

    PubMed Central

    Collins, P L; Hill, M G; Cristina, J; Grosfeld, H

    1996-01-01

    RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions. Images Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:8552680

  16. Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis

    PubMed Central

    Stoianov, Alexandra M.; Robson, Debra L.; Hetherington, Alexandra M.; Sawyez, Cynthia G.; Borradaile, Nica M.

    2015-01-01

    Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity. PMID:26102086

  17. The translation elongation factor eEF1A1 couples transcription to translation during heat shock response.

    PubMed

    Vera, Maria; Pani, Bibhusita; Griffiths, Lowri A; Muchardt, Christian; Abbott, Catherine M; Singer, Robert H; Nudler, Evgeny

    2014-09-16

    Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3'UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.

  18. A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

    PubMed Central

    Ruehle, Marisa D; Zhang, Haibo; Sheridan, Ryan M; Mitra, Somdeb; Chen, Yuanwei; Gonzalez, Ruben L; Cooperman, Barry S; Kieft, Jeffrey S

    2015-01-01

    Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs. DOI: http://dx.doi.org/10.7554/eLife.08146.001 PMID:26523395

  19. West syndrome caused by homozygous variant in the evolutionary conserved gene encoding the mitochondrial elongation factor GUF1.

    PubMed

    Alfaiz, Ali Abdullah; Müller, Verena; Boutry-Kryza, Nadia; Ville, Dorothée; Guex, Nicolas; de Bellescize, Julitta; Rivier, Clotilde; Labalme, Audrey; des Portes, Vincent; Edery, Patrick; Till, Marianne; Xenarios, Ioannis; Sanlaville, Damien; Herrmann, Johannes M; Lesca, Gaétan; Reymond, Alexandre

    2016-07-01

    West syndrome (WS), defined by the triad of infantile spasms, pathognomonic hypsarrhythmia and developmental regression, is a rare epileptic disease affecting about 1:3500 live births. To get better insights on the genetic of this pathology, we exome-sequenced the members of a consanguineous family affected with isolated WS. We identified a homozygous variant (c.1825G>T/p.(Ala609Ser)) in the GUF1 gene in the three affected siblings. GUF1 encodes a protein essential in conditions that counteract faithful protein synthesis: it is able to remobilize stuck ribosomes and transiently inhibit the elongation process to optimize protein synthesis. The variant identified in the WS family changes an alanine residue conserved in all eukaryotic organisms and positioned within the tRNA-binding moiety of this nuclear genome-encoded mitochondrial translational elongation factor. Yeast complementation assays show that the activity of GUF1(A609S) is modified in suboptimal environments. We suggest a new link between improper assembly of respiratory chain complexes and WS.

  20. Binding of Tat to TAR and Recruitment of Positive Transcription Elongation Factor b Occur Independently in Bovine Immunodeficiency Virus

    PubMed Central

    Barboric, Matjaz; Taube, Ran; Nekrep, Nada; Fujinaga, Koh; Peterlin, B. Matija

    2000-01-01

    Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5′ bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV. PMID:10846086

  1. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

    PubMed Central

    Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D.

    2016-01-01

    The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt-) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt- mutants. We determine that the lys2-128∂ Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt- transcription in tested Spt- mutants. PMID:27261007

  2. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae.

    PubMed

    Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D

    2016-08-09

    The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt(-)) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS(+) phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt(-) phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6 We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt(-) mutants. We determine that the lys2-128∂ Spt(-) phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt(-) transcription in tested Spt(-) mutants.

  3. Crystal structures of the human elongation factor eEFSec suggest a non-canonical mechanism for selenocysteine incorporation

    PubMed Central

    Dobosz-Bartoszek, Malgorzata; Pinkerton, Mark H.; Otwinowski, Zbyszek; Chakravarthy, Srinivas; Söll, Dieter; Copeland, Paul R.; Simonović, Miljan

    2016-01-01

    Selenocysteine is the only proteinogenic amino acid encoded by a recoded in-frame UGA codon that does not operate as the canonical opal stop codon. A specialized translation elongation factor, eEFSec in eukaryotes and SelB in prokaryotes, promotes selenocysteine incorporation into selenoproteins by a still poorly understood mechanism. Our structural and biochemical results reveal that four domains of human eEFSec fold into a chalice-like structure that has similar binding affinities for GDP, GTP and other guanine nucleotides. Surprisingly, unlike in eEF1A and EF-Tu, the guanine nucleotide exchange does not cause a major conformational change in domain 1 of eEFSec, but instead induces a swing of domain 4. We propose that eEFSec employs a non-canonical mechanism involving the distinct C-terminal domain 4 for the release of the selenocysteinyl-tRNA during decoding on the ribosome. PMID:27708257

  4. Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human.

    PubMed

    Hamey, Joshua J; Winter, Daniel L; Yagoub, Daniel; Overall, Christopher M; Hart-Smith, Gene; Wilkins, Marc R

    2016-01-01

    Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys(79) in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys(79) methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.

  5. Role of yeast peptide elongation factor 3 (EF-3) at the AA-tRNA binding step.

    PubMed

    Uritani, M; Miyazaki, M

    1988-07-01

    The stimulatory effect of peptide elongation factor 3 (EF-3), which is uniquely required for the yeast elongation cycle, on the step of binding of aminoacyl-tRNA (AA-tRNA) to ribosomes has been investigated in detail. Yeast EF-1 alpha apparently functions in a stoichiometric manner in the binding reaction of AA-tRNA to the ribosomes. The addition of EF-3 and ATP to this binding system strikingly stimulated the binding reaction, and the stimulated reaction proceeded catalytically with respect to both EF-1 alpha and EF-3, accompanied by ATP hydrolysis, indicating that EF-3 stimulated the AA-tRNA binding reaction by releasing EF-1 alpha from the ribosomal complex, thus recycling it. This binding stimulation by EF-3 was in many respects distinct from that by EF-1 beta gamma. The idea that EF-3 may participate in the regeneration of GTP from ATP and the formed GDP, as indicated by the findings that the addition of EF-3 along with ATP allowed the AA-tRNA binding and Phe polymerization reactions to proceed even in the presence of GDP in place of GTP, was not verified by the results of direct measurement of [32P]GTP formation from [gamma-32P]ATP and GDP under various conditions. Examination of the stability of the bound AA-tRNA disclosed the different binding states of AA-tRNA on ribosomes between in the cases of the complexes formed with EF-1 alpha alone, or factor-independently, and with EF-1 alpha and EF-3.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Exploratory factor analysis for differentiating sensory and mechanical variables related to muscle-tendon unit elongation

    PubMed Central

    Chagas, Mauro H.; Magalhães, Fabrício A.; Peixoto, Gustavo H. C.; Pereira, Beatriz M.; Andrade, André G. P.; Menzel, Hans-Joachim K.

    2016-01-01

    ABSTRACT Background Stretching exercises are able to promote adaptations in the muscle-tendon unit (MTU), which can be tested through physiological and biomechanical variables. Identifying the key variables in MTU adaptations is crucial to improvements in training. Objective To perform an exploratory factor analysis (EFA) involving the variables often used to evaluate the response of the MTU to stretching exercises. Method Maximum joint range of motion (ROMMAX), ROM at first sensation of stretching (FSTROM), peak torque (torqueMAX), passive stiffness, normalized stiffness, passive energy, and normalized energy were investigated in 36 participants during passive knee extension on an isokinetic dynamometer. Stiffness and energy values were normalized by the muscle cross-sectional area and their passive mode assured by monitoring the EMG activity. Results EFA revealed two major factors that explained 89.68% of the total variance: 53.13% was explained by the variables torqueMAX, passive stiffness, normalized stiffness, passive energy, and normalized energy, whereas the remaining 36.55% was explained by the variables ROMMAX and FSTROM. Conclusion This result supports the literature wherein two main hypotheses (mechanical and sensory theories) have been suggested to describe the adaptations of the MTU to stretching exercises. Contrary to some studies, in the present investigation torqueMAX was significantly correlated with the variables of the mechanical theory rather than those of the sensory theory. Therefore, a new approach was proposed to explain the behavior of the torqueMAX during stretching exercises. PMID:27437715

  7. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

  8. The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN

    PubMed Central

    Shine, M.B.; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

    2016-01-01

    The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection. PMID:27356973

  9. Divergent Protein Motifs Direct Elongation Factor P-Mediated Translational Regulation in Salmonella enterica and Escherichia coli

    PubMed Central

    Hersch, Steven J.; Wang, Mengchi; Zou, S. Betty; Moon, Kyung-Mee; Foster, Leonard J.; Ibba, Michael; Navarre, William Wiley

    2013-01-01

    ABSTRACT Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation. PMID:23611909

  10. Determination of occupancies of the SPH and GT-IIC transcription factor binding motifs in SV40: evidence for two forms of transcription elongation complex.

    PubMed

    Eadara, J K; Lutter, L C

    1996-09-01

    Occupancies of the SPH and GT-IIC sequence motifs in the native SV40 late transcription elongation complex were determined by assessing blockage to restriction enzyme cleavage. Cleavages specific to the transcription elongation complex were quantified by radioactive extension labeling and polymerase run-off analysis. The SPH motif was assayed by Sphl digestion and found to be unoccupied. In contrast, digestion with Pvull at the GT-IIC site was blocked in 36% of the complexes, indicating that approximately a third of the complexes are occupied by factor. This fractional occupancy indicates that there are at least two forms of SV40 late transcription elongation complexes, one form with the GT-IIC site occupied by a factor and another with the site vacant.

  11. Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis.

    PubMed

    Laibach, Natalie; Hillebrand, Andrea; Twyman, Richard M; Prüfer, Dirk; Schulze Gronover, Christian

    2015-05-01

    Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis-prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF-silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF-silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production. PMID:25809497

  12. In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha).

    PubMed

    Gruber, A D; Levine, R A

    1997-05-01

    Elongation factor-1 alpha (EF-1 alpha) is an evolutionarily highly conserved universal cofactor of protein synthesis in all living cells. In this study, its use as a positive control in situ hybridization assays for specific detection of mRNA sequences was evaluated. Northern blot analysis of various non-neoplastic and neoplastic cultured cells of different stages of confluence, cell shape, and cell cycle status revealed that EF-1 alpha had a lower and more homogeneous expression than did beta-actin. In situ hybridization assays using digoxigenin-labeled riboprobes for the detection of EF-1 alpha mRNA in routinely formalin-fixed, paraffin-embedded tissue sections showed that EF-1 alpha is a suitable positive control in all types of cells. However, variation of protease pretreatments demonstrated distinct and sometimes mutually exclusive digestion conditions for different cell types within the same tissue sample. Our results indicate that detection of EF-1 alpha mRNA is an appropriate internal standard for in situ hybridization assays and that it is useful to control artifacts such as false negatives caused by inappropriate protease pretreatments. The observed variability of optimal protease pretreatments for different cell types within the same tissue section strengthens the importance of a positive control in in situ hybridization assays.

  13. The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

    SciTech Connect

    Yoshino, Tomoko; Maeda, Yasuo; Amagai, Aiko . E-mail: aiamagai@mail.tains.tohoku.ac.jp

    2007-08-03

    Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method. Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.

  14. Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis.

    PubMed

    Laibach, Natalie; Hillebrand, Andrea; Twyman, Richard M; Prüfer, Dirk; Schulze Gronover, Christian

    2015-05-01

    Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis-prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF-silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF-silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production.

  15. The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae).

    PubMed

    Díaz, Sebastián; Triana-Chávez, Omar; Gómez-Palacio, Andrés

    2016-09-01

    Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera.

  16. Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P

    PubMed Central

    Peil, Lauri; Starosta, Agata L.; Lassak, Jürgen; Atkinson, Gemma C.; Virumäe, Kai; Spitzer, Michaela; Tenson, Tanel; Jung, Kirsten; Remme, Jaanus; Wilson, Daniel N.

    2013-01-01

    Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation. PMID:24003132

  17. Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae.

    PubMed

    Sandbaken, M G; Culbertson, M R

    1988-12-01

    A mutational analysis of the eukaryotic elongation factor EF-1 alpha indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1 alpha. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1 alpha in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1 alpha protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1 alpha. The identification of frameshift and nonsense suppressor mutations in EF-1 alpha indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms. PMID:3066688

  18. The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae).

    PubMed

    Díaz, Sebastián; Triana-Chávez, Omar; Gómez-Palacio, Andrés

    2016-09-01

    Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera. PMID:27268149

  19. RNA interference against transcription elongation factor SII does not support its role in transcription-coupled nucleotide excision repair.

    PubMed

    Mackinnon-Roy, Christine; Stubbert, Lawton J; McKay, Bruce C

    2011-01-10

    RNA polymerase II is unable to bypass bulky DNA lesions induced by agents like ultraviolet light (UV light) and cisplatin that are located in the template strand of active genes. Arrested polymerases form a stable ternary complex at the site of DNA damage that is thought to pose an impediment to the repair of these lesions. Transcription-coupled nucleotide excision repair (TC-NER) preferentially repairs these DNA lesions through an incompletely defined mechanism. Based on elegant in vitro experiments, it was hypothesized that the transcription elongation factor IIS (TFIIS) may be required to couple transcription to repair by catalyzing the reverse translocation of the arrested polymerase, allowing access of repair proteins to the site of DNA damage. However the role of TFIIS in this repair process has not been tested in vivo. Here, silencing TFIIS using an RNA interference strategy did not affect the ability of cells to recover nascent RNA synthesis following UV exposure or the ability of cells to repair a UV-damaged reporter gene while a similar strategy to decrease the expression Cockayne syndrome group B protein (CSB) resulted in the expected repair defect. Furthermore, RNA interference against TFIIS did not increase the sensitivity of cells to UV light or cisplatin while decreased expression of CSB did. Taken together, these results indicate that TFIIS is not limiting for the repair of transcription-blocking DNA lesions and thus the present work does not support a role for TFIIS in TC-NER.

  20. Synthesis of Elongated Microcapsules

    NASA Technical Reports Server (NTRS)

    Li, Wenyan; Buhrow, Jerry; Calle, Luz M.

    2011-01-01

    One of the factors that influence the effectiveness of self-healing in functional materials is the amount of liquid healing agents that can be delivered to the damaged area. The use of hollow tubes or fibers and the more sophisticated micro-vascular networks has been proposed as a way to increase the amount of healing agents that can be released when damage is inflicted. Although these systems might be effective in some specific applications, they are not practical for coatings applications. One possible practical way to increase the healing efficiency is to use microcapsules with high-aspect-ratios, or elongated microcapsules. It is understood that elongated microcapsules will be more efficient because they can release more healing agent than a spherical microcapsule when a crack is initiated in the coating. Although the potential advantage of using elongated microcapsules for self healing applications is clear, it is very difficult to make elongated microcapsules from an emulsion system because spherical microcapsules are normally formed due to the interfacial tension between the dispersed phase and the continuous phase. This paper describes the two methods that have been developed by the authors to synthesize elongated microcapsules. The first method involves the use of an emulsion with intermediate stability and the second involves the application of mechanical shear conditions to the emulsion.

  1. Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

  2. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

    PubMed Central

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M.; Denis, Clyde L.

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation. PMID:26953568

  3. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

    PubMed

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M; Denis, Clyde L

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation. PMID:26953568

  4. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

    PubMed

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M; Denis, Clyde L

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

  5. Functional Characterization of a Gene in Sedum alfredii Hance Resembling Rubber Elongation Factor Endowed with Functions Associated with Cadmium Tolerance.

    PubMed

    Liu, Mingying; Qiu, Wenming; He, Xuelian; Zheng, Liu; Song, Xixi; Han, Xiaojiao; Jiang, Jing; Qiao, Guirong; Sang, Jian; Liu, Mingqing; Zhuo, Renying

    2016-01-01

    Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the rubber elongation factor gene designated as SaREFl. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, SaREFl-expressing Arabidopsis plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that SaREFl responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of SaREFl as a bait led to the identification of five possible interacting targets in Sedum alfredii Hance. Among them, a gene annotated as prenylated Rab acceptor 1 (PRA1) domain protein was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants. PMID:27446189

  6. Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin

    PubMed Central

    Losada, Alejandro; Muñoz-Alonso, María José; García, Carolina; Sánchez-Murcia, Pedro A.; Martínez-Leal, Juan Fernando; Domínguez, Juan Manuel; Lillo, M. Pilar; Gago, Federico; Galmarini, Carlos M.

    2016-01-01

    eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a KD of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [14C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin. PMID:27713531

  7. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  8. Functional Characterization of a Gene in Sedum alfredii Hance Resembling Rubber Elongation Factor Endowed with Functions Associated with Cadmium Tolerance

    PubMed Central

    Liu, Mingying; Qiu, Wenming; He, Xuelian; Zheng, Liu; Song, Xixi; Han, Xiaojiao; Jiang, Jing; Qiao, Guirong; Sang, Jian; Liu, Mingqing; Zhuo, Renying

    2016-01-01

    Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the rubber elongation factor gene designated as SaREFl. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, SaREFl-expressing Arabidopsis plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that SaREFl responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of SaREFl as a bait led to the identification of five possible interacting targets in Sedum alfredii Hance. Among them, a gene annotated as prenylated Rab acceptor 1 (PRA1) domain protein was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants. PMID:27446189

  9. New insights on the interaction between the isoforms 1 and 2 of human translation elongation factor 1A.

    PubMed

    Migliaccio, Nunzia; Ruggiero, Immacolata; Martucci, Nicola M; Sanges, Carmen; Arbucci, Salvatore; Tatè, Rosarita; Rippa, Emilia; Arcari, Paolo; Lamberti, Annalisa

    2015-11-01

    The eukaryotic translation elongation factor 1A (eEF1A) is a moonlighting protein that besides to its canonical role in protein synthesis is also involved in many other cellular processes such as cell survival and apoptosis. In a previous work, we identified eEF1A Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and apoptosis of human cancer cells. We proposed that the phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms thus suggesting the formation of a potential eEF1A heterodimer owning regulatory properties. This study aimed at investigating the cellular localization and interaction between two eEF1A isoforms. To this end, we developed chimera proteins by adding at the N-terminal end of both eEF1A1 and eEF1A2 cyan fluorescence protein (mCerulean) and yellow fluorescence protein (mVenus), respectively. The fluorescent eEF1A1 and eEF1A2 chimeras were both addressed to COS-7 cells and found co-localized in the cytoplasm at the level of cellular membranes. We highlighted FRET between the labeled N-termini of eEF1A isoforms. The intra-molecular FRET of this chimera was about 17%. Our results provide novel information on the intracellular distribution and interaction of eEF1A isoforms.

  10. Everolimus enhances cellular cytotoxicity of lapatinib via the eukaryotic elongation factor-2 kinase pathway in nasopharyngeal carcinoma cells

    PubMed Central

    Liu, Lin; Wang, Zhi-Hui; Han, Jun; Tang, Con; Chen, Nan; Lin, Zhong; Peng, Pei-Jian

    2016-01-01

    Background Nasopharyngeal carcinoma (NPC) has a high relapse and metastatic rates; hence, development of new therapeutics is an immediate requirement. Lapatinib and everolimus have been demonstrated to be effective in the treatment of several carcinomas. This preclinical study aimed to investigate the effect and mechanism of lapatinib combined with everolimus on NPC cells. Methods The Cell Counting Kit 8 and colony formation assay were used to detect the effect of lapatinib alone or lapatinib combined with everolimus on the growth and proliferation of cells. Apoptosis was tested by flow cytometry and was further confirmed by western blot. The targets of lapatinib and the effects of lapatinib or everolimus on the eukaryotic elongation factor-2 (eEF-2) kinase pathway were analyzed by western blot, which also evaluated autophagy activity. Results Lapatinib inhibited the cellular viability and colony formation in NPC cells. At 24–72 h, the average half maximal inhibitory concentration (IC50) values of lapatinib were ranging from 3 to 5 μM. This study further found that lapatinib induced both apoptosis and autophagy in NPC cells, and this autophagic activity was described as type II programmed cell death via an eEF-2 kinase-dependent pathway. In addition, augmentation of lapatinib-induced autophagy by mammalian target of rapamycin (mTOR) inhibitor everolimus enhanced the cytocidal effect of lapatinib in NPC cells via the mTOR/S6 kinase/eEF-2 kinase pathway. Conclusion This study reveals that everolimus can sensitize NPC cells to lapatinib by the activation of eEF-2 kinase and provides a potential model of combination therapy. PMID:27785067

  11. Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

    PubMed

    Nagata, S; Tsunetsugu-Yokota, Y; Naito, A; Kaziro, Y

    1983-10-01

    A 3.1-kilobase Bgl II fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'- and 3'-flanking regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-1A transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-1 alpha. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu. PMID:6353412

  12. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  13. Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling

    PubMed Central

    Qiu, Fu-Nan; Huang, Yi; Chen, Dun-Yan; Li, Feng; Wu, Yan-An; Wu, Wen-Bing; Huang, Xiao-Li

    2016-01-01

    AIM: To assess the impact of eukaryotic elongation factor 1 alpha 2 (eEF1A2) on hepatocellular carcinoma (HCC) cell proliferation, apoptosis, migration and invasion, and determine the underlying mechanisms. METHODS: eEF1A2 levels were detected in 62 HCC tissue samples and paired pericarcinomatous specimens, and the human HCC cell lines SK-HEP-1, HepG2 and BEF-7402, by real-time PCR and immunohistochemistry. Experimental groups included eEF1A2 silencing in BEL-7402 cells with lentivirus eEF1A2-shRNA (KD group) and eEF1A2 overexpression in SK-HEP-1 cells with eEF1A2 plasmid (OE group). Non-transfected cells (control group) and lentivirus-based empty vector transfected cells (NC group) were considered control groups. Cell proliferation (MTT and colony formation assays), apoptosis (Annexin V-APC assay), cell cycle (DNA ploidy assay), and migration and invasion (Transwell assays) were assessed. Protein levels of PI3K/Akt/NF-κB signaling effectors were evaluated by Western blot. RESULTS: eEF1A2 mRNA and protein levels were significantly higher in HCC cancer tissue samples than in paired pericarcinomatous and normal specimens. SK-HEP-1 cells showed lower eEF1A2 mRNA levels; HepG2 and BEL-7402 cells showed higher eEF1A2 mRNA levels, with BEL-7402 cells displaying the highest amount. Efficient eEF1A2 silencing resulted in reduced cell proliferation, migration and invasion, increased apoptosis, and induced cell cycle arrest. The PI3K/Akt/NF-κB signaling pathway was notably inhibited. Inversely, eEF1A2 overexpression resulted in promoted cell proliferation, migration and invasion. CONCLUSION: eEF1A2, highly expressed in HCC, is a potential oncogene. Its silencing significantly decreases HCC tumorigenesis, likely by inhibiting PI3K/Akt/NF-κB signaling. PMID:27122673

  14. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA

    SciTech Connect

    Rasubala, Linda; Fourmy, Dominique; Ose, Toyoyuki; Kohda, Daisuke; Maenaka, Katsumi Yoshizawa, Satoko

    2005-03-01

    The mRNA-binding domain of M. thermoacetica selenocysteine-specific elongation factor SelB (residues 512–634, SelB-M) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was chemically prepared. The purified SelB-M–SECIS RNA complex has been crystallized in space group P2{sub 1}2{sub 1}2 and diffracted to 2.3 Å.

  15. Sequence analysis of the peptide-elongation factor EF-2 gene, downstream from those of ribosomal proteins H-S12 and H-S7, from the archaebacterial extreme halophile, Halobacterium halobium.

    PubMed

    Itoh, T

    1989-12-01

    The gene for the peptide-elongation factor 2 (EF-2) was cloned from the archaebacterial extreme halophile Halobacterium halobium and sequenced. The 1013 nucleotides upstream from this gene was two open reading frames similar to ribosomal proteins S12 and S7 from Escherichia coli. Sequence alignment studies showed the halobacterial elongation factor 2 to be equivalent to eukaryotic EF-2 and eubacterial EF-G. Sequence similarity to the eukaryotic elongation factor was much higher than to the eubacterial factor. Conserved sequence regions were present within the factor and are likely to constitute functionally important domains. These include the sites of GTP binding and ADP ribosylation by diphtheria toxin.

  16. The phylogenetic position of the pelobiont Mastigamoeba balamuthi based on sequences of rDNA and translation elongation factors EF-1alpha and EF-2.

    PubMed

    Arisue, Nobuko; Hashimot, Tetsuo; Lee, Jennifer A; Moore, Dorothy V; Gordon, Paul; Sensen, Christoph W; Gaasterland, Terry; Hasegawa, Masami; Müller, Miklós

    2002-01-01

    The taxonomic position and phylogenetic relationships of the Pelobionta, an amitochondriate amoeboflagellate group, are not yet completely settled. To provide more information, we obtained sequences for the large subunit rDNA gene, the gene for translation elongation factor 1alpha, and for a large part of the gene encoding translation elongation factor 2 from a representative of this group, Mastigamoeba balamuthi (formerly Phreatamoeba balamuthi). The gene for the large subunit rDNA was unusually large compared to those of other protists, a phenomenon that had previously been observed for the gene encoding the small subunit rDNA. Phylogenetic reconstruction using a maximum likelihood method was performed with these sequences, as well as the gene encoding the small subunit rDNA. When evaluated individually, the M. balamuthi genes for the small and large subunit rDNAs and elongation factor 1alpha had a most recent common ancestor with either the Mycetozoa (slime molds) or with Entamoeba histolytica. A clade formed by M. balamuthi, E. histolytica, and Mycetozoa was not rejected statistically for any of the sequences. A combined maximum likelihood analysis using 3,935 positions from all molecules suggested that these three taxonomic units form a robust clade. We were unable to resolve the closest group to this clade using the combined analysis. These findings support the notion, which had previously been proposed primarily on cytological evidence, that both M. balamuthi and E. histolytica are closely related to the Mycetozoa and that these three together represent a major eukaryotic lineage.

  17. Thermoperiodic Control of Hypocotyl Elongation Depends on Auxin-Induced Ethylene Signaling That Controls Downstream PHYTOCHROME INTERACTING FACTOR3 Activity1

    PubMed Central

    Bours, Ralph; Kohlen, Wouter; Bouwmeester, Harro J.

    2015-01-01

    We show that antiphase light-temperature cycles (negative day-night temperature difference [−DIF]) inhibit hypocotyl growth in Arabidopsis (Arabidopsis thaliana). This is caused by reduced cell elongation during the cold photoperiod. Cell elongation in the basal part of the hypocotyl under −DIF was restored by both 1-aminocyclopropane-1-carboxylic acid (ACC; ethylene precursor) and auxin, indicating limited auxin and ethylene signaling under −DIF. Both auxin biosynthesis and auxin signaling were reduced during −DIF. In addition, expression of several ACC Synthase was reduced under −DIF but could be restored by auxin application. In contrast, the reduced hypocotyl elongation of ethylene biosynthesis and signaling mutants could not be complemented by auxin, indicating that auxin functions upstream of ethylene. The PHYTOCHROME INTERACTING FACTORS (PIFs) PIF3, PIF4, and PIF5 were previously shown to be important regulators of hypocotyl elongation. We now show that, in contrast to pif4 and pif5 mutants, the reduced hypocotyl length in pif3 cannot be rescued by either ACC or auxin. In line with this, treatment with ethylene or auxin inhibitors reduced hypocotyl elongation in PIF4 overexpressor (PIF4ox) and PIF5ox but not PIF3ox plants. PIF3 promoter activity was strongly reduced under −DIF but could be restored by auxin application in an ACC Synthase-dependent manner. Combined, these results show that PIF3 regulates hypocotyl length downstream, whereas PIF4 and PIF5 regulate hypocotyl length upstream of an auxin and ethylene cascade. We show that, under −DIF, lower auxin biosynthesis activity limits the signaling in this pathway, resulting in low activity of PIF3 and short hypocotyls. PMID:25516603

  18. The Rho guanine exchange factor RHGF-2 acts through the Rho-binding kinase LET-502 to mediate embryonic elongation in C. elegans.

    PubMed

    Chan, Benjamin G; Rocheleau, Simon K; Smit, Ryan B; Mains, Paul E

    2015-09-15

    Morphogenesis allows an organism to develop its final body shape. In Caenorhabditis elegans, a smooth muscle-like contraction of an actin/myosin network in the epidermis mediates the elongation of the worm embryo from a ball of cells into a long, thin worm. This process is controlled by two redundant pathways, one involving the small GTPase RHO-1 and its downstream effectors LET-502/Rho-binding kinase and MEL-11/myosin phosphatase, and another involving PAK-1/p21 activated kinase and FEM-2/PP2c phosphatase. Contraction occurs primarily in the lateral epidermal cells during elongation while the dorsal and ventral epidermal cells have a more passive role, and localized activity of a Rho GEF (guanine exchange factor) could contribute to this asymmetry. We found that loss of the C. elegans Rho GEF encoded by rhgf-2 results in arrest during early elongation. Genetically, rhgf-2 acts as an activator of let-502/Rho-binding kinase, in parallel to fem-2/PP2c phosphatase. Although expressed throughout the embryo, lateral cell-specific RHGF-2 expression can mediate elongation. The Rho GTPase activating protein (GAP) RGA-2 is known to inhibit contraction in the dorsal and ventral epidermis. Although rhgf-2 and rga-2 are individually lethal, the double mutant is viable with elongation still occurring in a let-502 dependent fashion. This indicates that LET-502/Rho-binding kinase has activity independent of the GEF and GAP. Finally, maternal LET-502 and MEL-11 are known to regulate the rate of cleavage furrow ingression in the early embryo and we show that maternal RHGF-2 also influences cleavage but RGA-2 does not. Thus while the LET-502/MEL-11 pathway is employed multiple times during embryogenesis, regulation by GEFs and GAPs differs at different points of the life cycle and fine tunes contractile function.

  19. Dictyostelium ribosomal protein genes and the elongation factor 1B gene show coordinate developmental regulation which is under post-transcriptional control.

    PubMed

    Agarwal, A K; Blumberg, D D

    1999-06-01

    Starvation for amino acids initiates the developmental program in the cellular slime mold, Dictyostelium discoideum [19, 20]. One of the earliest developmental events is the decline in ribosomal protein synthesis [2, 17, 29, 30]. The ribosomal protein mRNAs are excluded from polysomes with 20 min to 1 h following the removal of nutrients, and their mRNA levels decline sharply at about 9 h into the 24-h developmental cycle [28, 31, 35, 36]. It has been generally assumed that the decline in r-protein mRNA levels during late development reflected a decline in the transcription rate [12, 32, 43]. Here we demonstrate that this is not the case. The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium. Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2%-0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4%-0.6% of the rRNA rate. This low but constant transcription rate is in contrast to a spore coat protein gene Psp D, which is transcribed at 6% of the rRNA rate in late developing cells. The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation. Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional [27]. PMID:10374261

  20. Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Budachetri, Khemraj; Ribeiro, José M. C.; Karim, Shahid

    2013-01-01

    Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex. PMID:24282621

  1. GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites.

    PubMed

    Valencia, A; Kjeldgaard, M; Pai, E F; Sander, C

    1991-06-15

    GTPase domains are functional and structural units employed as molecular switches in a variety of important cellular functions, such as growth control, protein biosynthesis, and membrane traffic. Amino acid sequences of more than 100 members of different subfamilies are known, but crystal structures of only mammalian ras p21 and bacterial elongation factor Tu have been determined. After optimal superposition of these remarkably similar structures, careful multiple sequence alignment, and calculation of residue-residue interactions, we analyzed the two subfamilies in terms of structural conservation, sequence conservation, and residue contact strength. There are three main results. (i) A structure-based alignment of p21 and elongation factor Tu. (ii) The definition of a common conserved structural core that may be useful as the basis of model building by homology of the three-dimensional structure of any GTPase domain. (iii) Identification of sequence regions, other than the effector loop and the nucleotide binding site, that may be involved in the functional cycle: they are loop L4, known to change conformation after GTP hydrolysis; helix alpha 2, especially Arg-73 and Met-67 in ras p21; loops L8 and L10, including ras p21 Arg-123, Lys-147, and Leu-120; and residues located spatially near the N and C termini. These regions are candidate sites for interaction either with the GTP/GDP exchange factor, with a GTPase-affected function, or with a molecule delivered to a destination site with the aid of the GTPase domain.

  2. Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

    PubMed

    Blythe, Amanda; Gunasekara, Sanjika; Walshe, James; Mackay, Joel P; Hartzog, Grant A; Vrielink, Alice

    2014-08-01

    Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.

  3. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

    SciTech Connect

    Mann, Melanie C. Strobel, Sarah Fleckenstein, Bernhard Kress, Andrea K.

    2014-09-15

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo.

  4. The magic spot ppGpp influences in vitro the molecular and functional properties of the elongation factor 1α from the archaeon Sulfolobus solfataricus.

    PubMed

    Martucci, Nicola M; Lamberti, Anna; Vitagliano, Luigi; Cantiello, Piergiuseppe; Ruggiero, Immacolata; Arcari, Paolo; Masullo, Mariorosario

    2012-09-01

    Guanosine tetra-phosphate (ppGpp), also known as "magic spot I", is a key molecule in the stringent control of most eubacteria and some eukarya. Here, we show that ppGpp affects the functional and molecular properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α). Indeed, ppGpp inhibited archaeal protein synthesis in vitro, even though the concentration required to get inhibition was higher than that required for the eubacterial and eukaryal systems. Regarding the partial reactions catalysed by SsEF-1α the effect produced by ppGpp on the affinity for aa-tRNA was lower than that measured in the presence of GTP but higher than that for GDP. Magic spot I was also able to bind SsEF-1α with an intermediate affinity in comparison to that displayed by GDP and GTP. Furthermore, ppGpp inhibited the intrinsic GTPase of SsEF-1α with a competitive behaviour. Finally, the binding of ppGpp to SsEF-1α rendered the elongation factor more resistant to heat treatment and the analysis of the molecular model of the complex between SsEF-1α and ppGpp suggests that this stabilisation arises from the charge optimisation on the surface of the protein.

  5. The human mitochondrial elongation factor tu (EF-Tu) gene: cDNA sequence, genomic localization, genomic structure, and identification of a pseudogene.

    PubMed

    Ling, M; Merante, F; Chen, H S; Duff, C; Duncan, A M; Robinson, B H

    1997-09-15

    The human mitochondrial elongation factor Tu (EF-Tu) is nuclear-encoded and functions in the translational apparatus of mitochondria. The complete human EF-Tu cDNA sequence of 1677 base pairs (bp) with a 101 bp 5'-untranslated region, a 1368 bp coding region, and a 207 bp 3'-untranslated region, has been determined and updated. The predicted protein from this cDNA sequence is approximately 49.8 kDa in size and is composed of 455 amino acids (aa) with a putative N-terminal mitochondrial leader sequence of approximately 50 aa residues. The predicted amino acid sequence shows high similarity to other EF-Tu protein sequences from ox, yeast, and bacteria, and also shows limited similarity to human cystolic elongation factor 1 alpha. The complete size of this cDNA (1677 bp) obtained by cloning and sequencing was confirmed by Northern blot analysis, which showed a single transcript (mRNA) of approximately 1.7 kb in human liver. The genomic structure of this EF-Tu gene has been determined for the first time. This gene contains nine introns with a predicted size of approximately 3.6 kilobases (kb) and has been mapped to chromosome 16p11.2. In addition, an intronless pseudogene of approximately 1.7 kb with 92.6% nucleotide sequence similarity to the EF-Tu gene has also been identified and mapped to chromosome 17q11.2. PMID:9332382

  6. The transcription elongation factor NusA is required for stress-induced mutagenesis in Escherichia coli.

    PubMed

    Cohen, Susan E; Walker, Graham C

    2010-01-12

    Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac(+) revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac(+) revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis. PMID:20036541

  7. The Ability of Positive Transcription Elongation Factor b To Transactivate Human Immunodeficiency Virus Transcription Depends on a Functional Kinase Domain, Cyclin T1, and Tat

    PubMed Central

    Fujinaga, Koh; Cujec, Thomas P.; Peng, Junmin; Garriga, Judit; Price, David H.; Graña, Xavier; Peterlin, B. Matija

    1998-01-01

    By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat–P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome. PMID:9696809

  8. Localization of S1 and elongation factor-1 alpha mRNA in rat brain and liver by non-radioactive in situ hybridization.

    PubMed

    Lee, S; Stollar, E; Wang, E

    1993-07-01

    Elongation factor-1 alpha (EF-1 alpha) is a ubiquitous, highly conserved protein that functions in peptide elongation during mRNA translation. We recently reported that, as do lower species, mammals also contain a second EF-1 alpha-like gene (S1). Unlike EF-1 alpha, which is present in all tissues, S1 mRNA is detected only in brain, heart, and muscle by Northern analysis and RNAse protection assays. In this report we present the identification of S1 and EF-1 alpha messages by non-radioactive in situ hybridization in brain and liver. We show that with this technique we can detected S1 mRNA only in certain cells in brain, mostly neurons; on the other hand, EF-1 alpha is present in all cell types that we have studied so far. We demonstrate that although EF-1 alpha mRNA can be detected in S1-negative cells it is also present in high abundance in S1-positive cells. The results presented here correlate with our previous finding that mammalian species contain a tissue-specific EF-1 alpha-like gene, S1. The presence of a second EF-1 alpha-like transcript within fully differentiated cells suggests a novel cell type-specific gene expression whose function may be related to the permanent growth-arrested state of cells in brain, heart, and muscle. PMID:8515051

  9. Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

    PubMed

    Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

    2014-11-10

    Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

  10. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1

    PubMed Central

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants. PMID:26020533

  11. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1.

    PubMed

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants.

  12. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1.

    PubMed

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants. PMID:26020533

  13. The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones

    PubMed Central

    Truttmann, Matthias C.; Guo, Xuanzong; Engert, Christoph; Schwartz, Thomas U.; Ploegh, Hidde L.

    2016-01-01

    Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans. PMID:27138431

  14. What doesn't kill them makes them stronger: an association between elongation factor 1-α overdominance in the sea star Pisaster ochraceus and "sea star wasting disease".

    PubMed

    Wares, John P; Schiebelhut, Lauren M

    2016-01-01

    In recent years, a massive mortality event has killed millions of sea stars, of many different species, along the Pacific coast of North America. This disease event, known as 'sea star wasting disease' (SSWD), is linked to viral infection. In one affected sea star (Pisaster ochraceus), previous work had identified that the elongation factor 1-α locus (EF1A) harbored an intronic insertion allele that is lethal when homozygous yet appears to be maintained at moderate frequency in populations through increased fitness for heterozygotes. The environmental conditions supporting this increased fitness are unknown, but overdominance is often associated with disease. Here, we evaluate populations of P. ochraceus to identify the relationship between SSWD and EF1A genotype. Our data suggest that there may be significantly decreased occurrence of SSWD in individuals that are heterozygous at this locus. These results suggest further studies are warranted to understand the functional relationship between diversity at EF1A and survival in P. ochraceus.

  15. Microsatellite polymorphism in the promoter sequence of the elongation factor 3 gene of Candida albicans as the basis for a typing system.

    PubMed Central

    Bretagne, S; Costa, J M; Besmond, C; Carsique, R; Calderone, R

    1997-01-01

    The polymorphism of a TTC/TTTC microsatellite in the promoter sequence of the elongation factor 3 gene of Candida albicans was investigated by PCR. One primer was fluorescein labeled, and PCR signals were read with an automatic sequencer. Twenty-nine reference strains and 31 independent clinical isolates were studied. Eleven different alleles were identified, giving 16 different profiles among the 60 strains tested, with a discriminatory power of 0.88. This marker is stable upon subculture, and reproducibility was achieved by automated procedures. When several microsatellite markers are available, many isolates can be rapidly and reproducibly tested for epidemiological questions, such as the prevalence of a given strain in a hospital setting and transmission between patients. PMID:9196192

  16. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA

    SciTech Connect

    Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

    2007-05-01

    The mRNA-binding domain of E. coli selenocysteine-specific elongation factor SelB (residues 478–614; SelB-WH3/4) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was prepared by in vitro transcription. The purified SelB-WH3/4–SECIS RNA complex crystallized in space group C2 and diffracted to 2.3 Å. In bacteria, selenocysteine (the 21st amino acid) is incorporated into proteins via machinery that includes SelB, a specific translational elongation factor. SelB binds to an mRNA hairpin called the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA{sup Sec} to the ribosomal A site. The minimum C-terminal fragment (residues 478–614) of Escherichia coli SelB (SelB-WH3/4) required for SECIS binding has been overexpressed and purified. This protein was crystallized in complex with 23 nucleotides of the SECIS hairpin at 294 K using the hanging-drop vapour-diffusion method. A data set was collected to 2.3 Å resolution from a single crystal at 100 K using ESRF beamline BM-30. The crystal belongs to space group C2, with unit-cell parameters a = 103.50, b = 56.51, c = 48.41 Å. The asymmetric unit contains one WH3/4-domain–RNA complex. The Matthews coefficient was calculated to be 3.37 Å{sup 3} Da{sup −1} and the solvent content was estimated to be 67.4%.

  17. Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

    PubMed Central

    Jakobsson, Magnus E.; Davydova, Erna; Małecki, Jędrzej; Moen, Anders; Falnes, Pål Ø.

    2015-01-01

    The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6). PMID:26115316

  18. Accelerated evolution of functional plastid rRNA and elongation factor genes due to reduced protein synthetic load after the loss of photosynthesis in the chlorophyte alga Polytoma.

    PubMed

    Vernon, D; Gutell, R R; Cannone, J J; Rumpf, R W; Birky, C W

    2001-09-01

    Polytoma obtusum and Polytoma uvella are members of a clade of nonphotosynthetic chlorophyte algae closely related to Chlamydomonas humicola and other photosynthetic members of the Chlamydomonadaceae. Descended from a nonphotosynthetic mutant, these obligate heterotrophs retain a plastid (leucoplast) with a functional protein synthetic system, and a plastid genome (lpDNA) with functional genes encoding proteins required for transcription and translation. Comparative studies of the evolution of genes in chloroplasts and leucoplasts can identify modes of selection acting on the plastid genome. Two plastid genes--rrn16, encoding the plastid small-subunit rRNA, and tufA, encoding elongation factor Tu--retain their functions in protein synthesis after the loss of photosynthesis in two nonphotosynthetic Polytoma clades but show a substantially accelerated rate of base substitution in the P. uvella clade. The accelerated evolution of tufA is due, at least partly, to relaxed codon bias favoring codons that can be read without wobble, mainly in three amino acids. Selection for these codons may be relaxed because leucoplasts are required to synthesize fewer protein molecules per unit time than are chloroplasts (reduced protein synthetic load) and thus require a lower rate of synthesis of elongation factor Tu. Relaxed selection due to a lower protein synthetic load is also a plausible explanation for the accelerated rate of evolution of rrn16, but the available data are insufficient to test the hypothesis for this gene. The tufA and rrn16 genes in Polytoma oviforme, the sole member of a second nonphotosynthetic clade, are also functional but show no sign of relaxed selection.

  19. Methylation of translation elongation factor 1A by the METTL10-like See1 methyltransferase facilitates tombusvirus replication in yeast and plants.

    PubMed

    Li, Zhenghe; Gonzalez, Paulina Alatriste; Sasvari, Zsuzsanna; Kinzy, Terri Goss; Nagy, Peter D

    2014-01-01

    Replication of tombusviruses and other plus-strand RNA viruses depends on several host factors that are recruited into viral replicase complexes. Previous studies have shown that eukaryotic translation elongation factor 1A (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. In this paper, we show that methylation of eEF1A by the METTL10-like See1p methyltransferase is required for tombusvirus and unrelated nodavirus RNA replication in yeast model host. Similar to the effect of SEE1 deletion, yeast expressing only a mutant form of eEF1A lacking the 4 known lysines subjected to methylation supported reduced TBSV accumulation. We show that the half-life of several viral replication proteins is decreased in see1Δ yeast or when a mutated eEF1A was expressed as a sole source for eEF1A. Silencing of the plant ortholog of See1 methyltransferase also decreased tombusvirus RNA accumulation in Nicotiana benthamiana. PMID:24314635

  20. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    PubMed

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

  1. Cis and trans-acting elements involved in the activation of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha.

    PubMed Central

    Curie, C; Liboz, T; Bardet, C; Gander, E; Médale, C; Axelos, M; Lescure, B

    1991-01-01

    In A. thaliana the translation elongation factor EF-1 alpha is encoded by a small multigenic family of four members (A1-A4). The A1 gene promoter has been dissected and examined in a transient expression system using the GUS reporter gene. Deletion analysis has shown that several elements are involved in the activation process. One cis-acting domain, the TEF 1 box, has been accurately mapped 100 bp upstream of the transcription initiation site. This domain is the target for trans-acting factors identified in nuclear extracts prepared from A. thaliana. Homologies are found between the TEF 1 box and sequences present at the same location within the A2, A3 and A4 promoters. This observation, together with those obtained from gel retardation assays performed using DNA fragments from the A4 promoter, suggest that the activation process mediated by the TEF 1 element is conserved among the A. thaliana EF-1 alpha genes. Analysis of nearly full length cDNA clones has shown that in addition to a single intron located within the coding region, the A1 gene contains a second intron located within the 5' non coding region. Such an intron is also present within the A2, A3 and A4 genes. This 5' intervening sequence appears to be essential to obtain a maximum GUS activity driven by the A1 gene promoter. Images PMID:1840652

  2. Carbon monoxide releasing molecule-2 CORM-2 represses global protein synthesis by inhibition of eukaryotic elongation factor eEF2.

    PubMed

    Schwer, Christian Ingo; Stoll, Patrick; Rospert, Sabine; Fitzke, Edith; Schallner, Nils; Bürkle, Hartmut; Schmidt, Rene; Humar, Matjaz

    2013-02-01

    Carbon monoxide (CO) is an endogenous gaseous transmitter that exerts antiproliferative effects in many cell types, but effects of CO on the translational machinery are not described. We examined the effects of the carbon monoxide releasing molecule-2 (CORM-2) on critical steps in translational signaling and global protein synthesis in pancreatic stellate cells (PSCs), the most prominent collagen-producing cells in the pancreas, whose activation is associated with pancreatic fibrosis. PSCs were isolated from rat pancreatic tissue and incubated with CORM-2. CORM-2 prevented the decrease in the phosphorylation of eukaryotic elongation factor 2 (eEF2) caused by serum. By contrast, the activation dependent phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1) was inhibited by CORM-2 treatment. The phosphorylation of eukaryotic initiation factor 2α (eIF2α) and eukaryotic initiation factor 4E (eIF4E) were not affected by CORM-2 treatment. In consequence, CORM-2 mediated eEF2 phosphorylation and inactivation of 4E-BP1 suppressed global protein synthesis. These observations were associated with inhibition of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling and increased intracellular calcium and cAMP levels. The CORM-2 mediated inhibition of protein synthesis resulted in downregulation of cyclin D1 and cyclin E expression, a subsequent decline in the phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and cell growth arrest at the G(0)/G(1) phase checkpoint of the cell cycle. Our results suggest the therapeutic application of CO releasing molecules such as CORM-2 for the treatment of fibrosis, inflammation, cancer, or other pathologic states associated with excessive protein synthesis or hyperproliferation. However, prolonged exogenous application of CO might also have negative effects on cellular protein homeostasis.

  3. UV-B-responsive association of the Arabidopsis bZIP transcription factor ELONGATED HYPOCOTYL5 with target genes, including its own promoter.

    PubMed

    Binkert, Melanie; Kozma-Bognár, László; Terecskei, Kata; De Veylder, Lieven; Nagy, Ferenc; Ulm, Roman

    2014-10-01

    In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/G(HY5)-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B.

  4. UV-B-Responsive Association of the Arabidopsis bZIP Transcription Factor ELONGATED HYPOCOTYL5 with Target Genes, Including Its Own Promoter[W][OPEN

    PubMed Central

    Binkert, Melanie; Kozma-Bognár, László; Terecskei, Kata; De Veylder, Lieven; Nagy, Ferenc; Ulm, Roman

    2014-01-01

    In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/GHY5-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B. PMID:25351492

  5. Phylogenetic position of symbiotic protist Dinenympha [correction of Dinemympha] exilis in the hindgut of the termite Reticulitermes speratus inferred from the protein phylogeny of elongation factor 1 alpha.

    PubMed

    Moriya, S; Ohkuma, M; Kudo, T

    1998-04-14

    The phylogenetic position of the symbiotic oxymonad Dinenympha exilis, found in the hindgut of the lower termite Reticulitermes speratus, was determined by analysis of translation elongation factor 1 alpha (EF-1 alpha). cDNA corresponding to a major part of the amino acid coding region of EF-1 alpha mRNA was amplified by the reverse transcription polymerase chain reaction (RT-PCR) method from total mRNA of termite hindgut microorganisms without cultivation. The product was cloned into a plasmid vector, pGEM-T, and the clones were isolated and sequenced. One of the EF-1 alpha clones isolated was assigned to the protist D. exilis by whole-cell in-situ hybridization using a specific oligonucleotide probe with enzymatic signal amplification. The deduced amino acid sequence was aligned with those of other eukaryotic and archaeabacterial EF-1 alpha s, and the phylogenetic relationships among early branching eukaryotes were inferred by using the distance matrix method and the maximum parsimony method. The phylogenetic analysis indicated that the D. exilis offshoot occurred before mitochondria-containing organisms and D. exilis branched out after the diplomonads clade. These results indicate that the oxymonad D. exilis is one of the early branching organisms and suggest that the oxymonads form a lineage independent of other early branching organisms. PMID:9573371

  6. Differential screening of a human pancreatic adenocarcinoma lambda gt11 expression library has identified increased transcription of elongation factor EF-1 alpha in tumour cells.

    PubMed

    Grant, A G; Flomen, R M; Tizard, M L; Grant, D A

    1992-03-12

    A human pancreatic adenocarcinoma lambda gt11 expression library was differentially screened with mRNA derived from normal and cancerous pancreatic tissues. Five clones preferentially hybridized with pancreatic adenocarcinoma mRNA. cDNA inserts from 4 of these clones were amplified by PCR, labelled with alpha 32P and used in Northern blot analysis against mRNA prepared from a variety of tumour and normal tissues. lambda GER-4 identified a pancreas-associated mRNA (greater than 10 kb) with no homology with known sequences at either the nucleic or amino-acid level. lambda GER-2 identified a 1.7-kb mRNA transcript that was over-expressed in mRNA prepared from pancreas, colon, breast, lung and gastric tumours relative to normal tissues. Sequence analysis and restriction-enzyme mapping showed that this clone was completely homologous with the active form of human elongation factor EF-1 alpha. This high level of EF-1 alpha-mRNA expression in tumour tissues lends support to the increasing evidence that EF-1 alpha is an important regulator of the cell cycle. PMID:1544708

  7. The nucleotide sequence of the gene coding for the elongation factor 1 alpha in Sulfolobus solfataricus. Homology of the product with related proteins.

    PubMed

    Arcari, P; Gallo, M; Ianniciello, G; Dello Russo, A; Bocchini, V

    1994-04-01

    The cloning and sequencing of the gene coding for the archaebacterial elongation factor 1 alpha (aEF-1 alpha) was performed by screening a Sulfolobus solfataricus genomic library using a probe constructed from the eptapeptide KNMITGA that is conserved in all the EF-1 alpha/EF-Tu known so far. The isolated recombinant phage contained the part of the aEF-1 alpha gene from amino acids 1 to 171. The other part (amino acids 162-435) was obtained through the amplification of the S. solfataricus DNA by PCR. The codon usage by the aEF-1 alpha gene showed a preference for triplets ending in A and/or T. This behavior was almost identical to that of the S. acidocaldarius EF-1 alpha gene but differed greatly from that of EF-1 alpha/EF-Tu genes in other archaebacteria eukaryotes and eubacteria. The translated protein is made of 435 amino acid residues and contains sequence motifs for the binding of GTP, tRNA and ribosome. Alignments of aEF-1 alpha with several EF-1 alpha/EF-Tu revealed that aEF-1 alpha is more similar to its eukaryotic than to its eubacterial counterparts. PMID:8148382

  8. Differential expression pattern of rubber elongation factor (REF) mRNA transcripts from high and low yielding clones of rubber tree (Hevea brasiliensis Muell. Arg.).

    PubMed

    Priya, P; Venkatachalam, P; Thulaseedharan, A

    2007-10-01

    In Hevea tree, rubber elongation factor (REF) is a key gene involved in rubber biosynthesis. Since the immaturity period for Hevea is 6 years, identification of molecular marker for latex yield potential will be beneficial for early selection of high yielding clones. The main objective of this research is to study the expression pattern of the REF gene in contrasting latex yield rubber clones (high and low yielding) by Northern blot as well as RT-PCR analysis. Accumulation of REF mRNA transcripts was significantly higher in latex cells compared to other cells of seedlings and mature Hevea trees. Northern results revealed that the level of REF gene expression in latex cells of high yielding rubber clones was significantly higher than in low yielders. According to RT-PCR results, the abundance of REF mRNA transcripts in latex cells was fivefold higher in the RRII105 clone, one of the most high yielding rubber clones. It is evident from the results that both tapping and ethephon treatment had a direct effect on induction of REF gene expression. Results demonstrate a positive correlation between REF gene expression pattern and latex yield.

  9. Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

    PubMed

    Jenkins, C; Fuerst, J A

    2001-05-01

    Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. PMID:11443344

  10. Assignment of human elongation factor 1{alpha} genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3

    SciTech Connect

    Lund, A.; Clark, B.; Knudsen, S.M.

    1996-09-01

    The human elongation factor 1 {alpha} gene family consists of at least 2 actively transcribed genes, EEF1A and EEF1A2, and more than 18 homologous loci. EEF1A2 is expressed ubiquitously, and both of them can function in translation. An EEF1A cDNA probe has previously been shown to cross-hybridize with several human chromosomes, but the location of the functional gene has not been established. We have mapped the functional EEF1A gene to 6q14 by combined fluorescence in situ hybridization (FISH) and PCR analysis of a somatic cell hybrid panel and mapped EEF1A2 to 20q13.3 by FISH. In addition, the 11 strongest cross-hybridizing loci (EEF1AL2-EEF1AL13) were mapped by FISH to 12p12, 9q34, 7p15-p21, 19q13, 3q26-q27, 7q33-q35, 1p13-p22, 2q12-q14, 5p12-q11, 1q31-q32, and Xq21. 17 refs., 1 fig., 1 tab.

  11. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax.

    PubMed

    Mann, Melanie C; Strobel, Sarah; Fleckenstein, Bernhard; Kress, Andrea K

    2014-09-01

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation.

  12. veA-dependent RNA-pol II transcription elongation factor like protein, RtfA, is associated with secondary metabolism and morphological development in Aspergillus nidulans

    PubMed Central

    Ramamoorthy, Vellaisamy; Shantappa, Sourabha; Dhingra, Sourabh; Calvo, Ana M.

    2012-01-01

    In Aspergillus nidulans the global regulatory gene veA is necessary for the biosynthesis of several secondary metabolites, including the mycotoxin sterigmatocystin (ST). In order to identify additional veA-dependent genetic elements involved in regulating ST production, we performed a mutagenesis on a deletion veA (ΔveA) strain to obtain revertant mutants (RM) that regained the capability to produce toxin. Genetic analysis and molecular characterization of one of the revertant mutants, RM3, revealed that a point mutation occurred at the coding region of the rtfA gene, encoding a RNA-pol II transcription elongation factor like protein, similar to Saccharomyces cerevisiae Rtf1. The A. nidulans rtfA gene product accumulates in nuclei. Deletion of rtfA gene in a ΔveA background restored mycotoxin production in a medium-dependent manner. rtfA also affects the production of other metabolites including penicillin. Biosynthesis of this antibiotic decreased in the absence of rtfA. Furthermore, rtfA is necessary for normal morphological development. Deletion of the rtfA gene in wild-type strains (veA+) resulted in a slight decrease in growth rate, drastic reduction in conidiation, and complete loss of sexual development. This is the first study of an Rtf1 like gene in filamentous fungi. We found rtfA putative orthologs extensively conserved in numerous fungal species. PMID:22783880

  13. BnEPFL6, an EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide gene, is required for filament elongation in Brassica napus.

    PubMed

    Huang, Yi; Tao, Zhangsheng; Liu, Qiong; Wang, Xinfa; Yu, Jingyin; Liu, Guihua; Wang, Hanzhong

    2014-07-01

    Inflorescence architecture, pedicel length and stomata patterning in Arabidopsis thaliana are specified by inter-tissue communication mediated by ERECTA and its signaling ligands in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family of secreted cysteine-rich peptides. Here, we identified and characterized BnEPFL6 from Brassica napus. Heterologous expression of this gene under the double enhanced CaMV promoter (D35S) in Arabidopsis resulted in shortened stamen filaments, filaments degradation, and reduced filament cell size that displayed down-regulated expression of AHK2, in which phenotypic variation of ahk2-1 mutant presented highly consistent with that of BnEPFL6 transgenic lines. Especially, the expression level of BnEPFL6 in the shortened filaments of four B. napus male sterile lines (98A, 86A, SA, and Z11A) was similar to that of BnEPFL6 in the transgenic Arabidopsis lines. The activity of pBnEPFL6.2::GUS was intensive in the filaments of transgenic lines. These observations reveal that BnEPFL6 plays an important role in filament elongation and may also affect organ morphology and floral organ specification via a BnEPFL6-mediated cascade. PMID:24838654

  14. Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage.

    PubMed

    Qiu, Lihua; Jiang, Shigui; Zhou, Falin; Zhang, Dianchang; Huang, Jianhua; Guo, Yihui

    2008-09-01

    The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.

  15. PSRP1 is not a ribosomal protein, but a ribosome-binding factor that is recycled by the ribosome-recycling factor (RRF) and elongation factor G (EF-G).

    PubMed

    Sharma, Manjuli R; Dönhöfer, Alexandra; Barat, Chandana; Marquez, Viter; Datta, Partha P; Fucini, Paola; Wilson, Daniel N; Agrawal, Rajendra K

    2010-02-01

    Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool. PMID:19965869

  16. A translation system reconstituted with human factors proves that processing of encephalomyocarditis virus proteins 2A and 2B occurs in the elongation phase of translation without eukaryotic release factors.

    PubMed

    Machida, Kodai; Mikami, Satoshi; Masutani, Mamiko; Mishima, Kurumi; Kobayashi, Tominari; Imataka, Hiroaki

    2014-11-14

    The genomic RNA of encephalomyocarditis virus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins 2A and 2B by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3 (eRF1 and eRF3), aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site. We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs. Our results indicate that this unusual event occurs in the elongation phase of translation.

  17. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    SciTech Connect

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  18. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2.

    PubMed

    Zhu, Qiuzhen; Zhang, Yuefan; Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  19. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

    PubMed Central

    Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  20. FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells

    PubMed Central

    Hamurcu, Zuhal; Ashour, Ahmed; Kahraman, Nermin; Ozpolat, Bulent

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K), an emerging molecular target for cancer therapy, contributes to cancer proliferation, cell survival, tumorigenesis, and invasion, disease progression and drug resistance. Although eEF2K is highly up-regulated in various cancers, the mechanism of gene regulation has not been elucidated. In this study, we examined the role of Forkhead Box M1 (FOXM1) proto-oncogenic transcription factor in triple negative breast cancer (TNBC) cells and the regulation of eEF2K. We found that FOXM1 is highly upregulated in TNBC and its knockdown by RNA interference (siRNA) significantly inhibited eEF2K expression and suppressed cell proliferation, colony formation, migration, invasion and induced apoptotic cell death, recapitulating the effects of eEF2K inhibition. Knockdown of FOXM1 inhibited regulators of cell cycle, migration/invasion and survival, including cyclin D1, Src and MAPK-ERK signaling pathways, respectively. We also demonstrated that FOXM1 (1B and 1C isoforms) directly binds to and transcriptionally regulates eEF2K gene expression by chromatin immunoprecipitation (ChIP) and luciferase gene reporter assays. Furthermore, in vivo inhibition of FOXM1 by liposomal siRNA-nanoparticles suppressed growth of MDA-MB-231 TNBC tumor xenografts in orthotopic models. In conclusion, our study provides the first evidence about the transcriptional regulation of eEF2K in TNBC and the role of FOXM1 in mediating breast cancer cell proliferation, survival, migration/invasion, progression and tumorgenesis and highlighting the potential of FOXM1/eEF2K axis as a molecular target in breast and other cancers. PMID:26918606

  1. Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2008-10-21

    NusA and NusG are transcription elongation factors that bind to RNA polymerase (RNAP) after sigma subunit release. Escherichia coli NusA (NusA(Ec)) stimulates intrinsic termination and RNAP(Ec) pausing, whereas NusG(Ec) promotes Rho-dependent termination and pause escape. Both Nus factors also participate in the formation of RNAP(Ec) antitermination complexes. We showed that Bacillus subtilis NusA (NusA(Bs)) stimulates intrinsic termination and RNAP(Bs) pausing at U107 and U144 in the trpEDCFBA operon leader. Pausing at U107 and U144 participates in the transcription attenuation and translational control mechanisms, respectively, presumably by providing additional time for trp RNA-binding attenuation protein (TRAP) to bind to the nascent trp leader transcript. Here, we show that NusG(Bs) causes modest pause stimulation at U107 and dramatic pause stimulation at U144. NusA(Bs) and NusG(Bs) act synergistically to increase the U107 and U144 pause half-lives. NusG(Bs)-stimulated pausing at U144 requires RNAP(Bs), whereas NusA(Bs) stimulates pausing of RNAP(Bs) and RNAP(Ec) at the U144 and E. coli his pause sites. Although NusG(Ec) does not stimulate pausing at U144, it competes with NusG(Bs)-stimulated pausing, suggesting that both proteins bind to the same surface of RNAP(Bs). Inactivation of nusG results in the loss of RNAP pausing at U144 in vivo and elevated trp operon expression, whereas plasmid-encoded NusG complements the mutant defects. Overexpression of nusG reduces trp operon expression to a larger extent than overexpression of nusA. PMID:18852477

  2. Elongation factor eEF1B modulates functions of the release factors eRF1 and eRF3 and the efficiency of translation termination in yeast

    PubMed Central

    Valouev, Igor A; Fominov, Gleb V; Sokolova, Elizaveta E; Smirnov, Vladimir N; Ter-Avanesyan, Michael D

    2009-01-01

    Background Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation. Results To characterize further the functions of yeast eRF1 and eRF3, a genetic screen for their novel partner proteins was performed. As a result, the genes for γ (TEF4 and TEF3/CAM1) and α (TEF5/EFB1) subunits of the translation elongation factor eEF1B, known to catalyze the exchange of bound GDP for GTP on eEF1A, were revealed. These genes act as dosage suppressors of a synthetic growth defect caused by some mutations in the SUP45 and SUP35 genes encoding eRF1 and eRF3, respectively. Extra copies of TEF5 and TEF3 can also suppress the temperature sensitivity of some sup45 and sup35 mutants and reduce nonsense codon readthrough caused by these omnipotent suppressors. Besides, overproduction of eEF1Bα reduces nonsense codon readthrough in the strain carrying suppressor tRNA. Such effects were not shown for extra copies of TEF2, which encodes eEF1A, thus indicating that they were not due to eEF1A activation. Conclusion The data obtained demonstrate involvement of the translation elongation factor eEF1B in modulating the functions of translation termination factors and suggest its possible role in GDP for GTP exchange on eRF3. PMID:19545407

  3. Molecular Phylogeny and Evolution of Parabasalia with Improved Taxon Sampling and New Protein Markers of Actin and Elongation Factor-1α

    PubMed Central

    Noda, Satoko; Mantini, Cléa; Meloni, Dionigia; Inoue, Jun-Ichi; Kitade, Osamu; Viscogliosi, Eric; Ohkuma, Moriya

    2012-01-01

    Background Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa—in particular determining the root—is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. Principal Findings Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. Conclusions/Significance We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in

  4. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

  5. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    PubMed Central

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  6. Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.

    PubMed

    Eckhardt, Katrin; Troger, Juliane; Reissmann, Jana; Katschinski, Dörthe M; Wagner, Klaus F; Stengel, Petra; Paasch, Uwe; Hunziker, Peter; Borter, Emanuela; Barth, Sandra; Schlafli, Philipp; Spielmann, Patrick; Stiehl, Daniel P; Camenisch, Gieri; Wenger, Roland H

    2007-01-01

    PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells. PMID:17595531

  7. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  8. Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

    PubMed Central

    Routh, Satya Brata; Ahmad, Sadeem; Suma, Katta; Kumar, Mantu; Kuncha, Santosh Kumar; Yadav, Kranthikumar; Kruparani, Shobha P; Sankaranarayanan, Rajan

    2016-01-01

    D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting. PMID:27224426

  9. Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification.

    PubMed

    Rosenberry, T L; Krall, J A; Dever, T E; Haas, R; Louvard, D; Merrick, W C

    1989-05-01

    Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine. PMID:2708357

  10. Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum *

    PubMed Central

    Matsubayashi, Makoto; Teramoto-Kimata, Isao; Uni, Shigehiko; Lillehoj, Hyun S.; Matsuda, Haruo; Furuya, Masaru; Tani, Hiroyuki; Sasai, Kazumi

    2013-01-01

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis. PMID:24085304

  11. In vitro and in vivo protection by melatonin against the decline of elongation factor-2 caused by lipid peroxidation: preservation of protein synthesis.

    PubMed

    Argüelles, Sandro; Muñoz, Mario F; Cano, Mercedes; Machado, Alberto; Ayala, Antonio

    2012-08-01

    As organisms age, a considerable decrease in protein synthesis takes place in all tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). eEF-2 occupies an essential role in protein synthesis where it catalyzes the ribosomal translocation reaction. eEF-2 is particularly sensitive to increased oxidative stress. However, all oxidants do not affect eEF-2, only compounds that increase lipid peroxidation. As peroxides are unstable compounds, they decompose and generate a series of highly reactive compounds, including aldehydes malondialdehyde (MDA) and 4-hydroxynoenal (HNE). We have previously reported that hepatic eEF-2 forms adducts with low-molecular weight aldehydes, MDA and HNE. Therefore, the protection of eEF-2 must be specifically carried out by a compound with lipoperoxyl radical-scavenging features such as melatonin. In this article, we show the ability of melatonin to protect against the changes that occur in the eEF-2 under conditions of lipid peroxidation induced by cumene hydroperoxide (CH), a compound used experimentally to induce lipid breakdown. As experimental models, we used cultured cells and rats treated with this oxidant compound. eEF-2 levels, adduct formation of this protein with MDA and HNE, and lipid peroxides were determined. In the cultured cells, protein synthesis rate was also measured. Our results show that melatonin prevented the molecular changes in eEF-2 and the decline in protein synthesis rate secondary to lipid peroxidation. The results also show that serum levels of several hormones were affected by CH-induced oxidative stress, which was partially or totally prevented by melatonin.

  12. Sulpiride, but not SCH23390, modifies cocaine-induced conditioned place preference and expression of tyrosine hydroxylase and elongation factor 1α in zebrafish.

    PubMed

    Darland, Tristan; Mauch, Justin T; Meier, Ellen M; Hagan, Shannon J; Dowling, John E; Darland, Diane C

    2012-12-01

    Finding genetic polymorphisms and mutations linked to addictive behavior can provide important targets for pharmaceutical and therapeutic interventions. Forward genetic approaches in model organisms such as zebrafish provide a potentially powerful avenue for finding new target genes. In order to validate this use of zebrafish, the molecular nature of its reward system must be characterized. We have previously reported the use of cocaine-induced conditioned place preference (CPP) as a reliable method for screening mutagenized fish for defects in the reward pathway. Here we test if CPP in zebrafish involves the dopaminergic system by co-treating fish with cocaine and dopaminergic antagonists. Sulpiride, a potent D2 receptor (DR2) antagonist, blocked cocaine-induced CPP, while the D1 receptor (DR1) antagonist SCH23390 had no effect. Acute cocaine exposure also induced a rise in the expression of tyrosine hydroxylase (TH), an important enzyme in dopamine synthesis, and a significant decrease in the expression of elongation factor 1α (EF1α), a housekeeping gene that regulates protein synthesis. Cocaine selectively increased the ratio of TH/EF1α in the telencephalon, but not in other brain regions. The cocaine-induced change in TH/EF1α was blocked by co-treatment with sulpiride, but not SCH23390, correlating closely with the action of these drugs on the CPP behavioral response. Immunohistochemical analysis revealed that the drop in EF1α was selective for the dorsal nucleus of the ventral telencephalic area (Vd), a region believed to be the teleost equivalent of the striatum. Examination of TH mRNA and EF1α transcripts suggests that regulation of expression is post-transcriptional, but this requires further examination. These results highlight important similarities and differences between zebrafish and more traditional mammalian model organisms. PMID:22910534

  13. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

  14. MicroRNA-33a-5p Modulates Japanese Encephalitis Virus Replication by Targeting Eukaryotic Translation Elongation Factor 1A1

    PubMed Central

    Chen, Zheng; Ye, Jing; Ashraf, Usama; Li, Yunchuan; Wei, Siqi; Wan, Shengfeng; Zohaib, Ali; Song, Yunfeng; Chen, Huanchun

    2016-01-01

    ABSTRACT Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis. IMPORTANCE MiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular mi

  15. Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly.

    PubMed

    Grozdanov, Petar N; MacDonald, Clinton C

    2015-02-09

    Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

  16. Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly.

    PubMed

    Grozdanov, Petar N; MacDonald, Clinton C

    2015-01-01

    Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification. PMID:25742071

  17. RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZ and Class-C bHLH Factors and Regulates Jasmonate-Induced Gene Expression and Root Cell Elongation[C][W

    PubMed Central

    Toda, Yosuke; Tanaka, Maiko; Ogawa, Daisuke; Kurata, Kyo; Kurotani, Ken-ichi; Habu, Yoshiki; Ando, Tsuyu; Sugimoto, Kazuhiko; Mitsuda, Nobutaka; Katoh, Etsuko; Abe, Kiyomi; Miyao, Akio; Hirochika, Hirohiko; Hattori, Tsukaho; Takeda, Shin

    2013-01-01

    Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions. PMID:23715469

  18. Elongated Microcapsules and Their Formation

    NASA Technical Reports Server (NTRS)

    Calle, Luz M. (Inventor); Li, Wenyan N. (Inventor); Buhrow, Jerry W. (Inventor); Perusich, Stephen A. (Inventor); Jolley, Scott T. (Inventor); Gibson, Tracy L. (Inventor); Williams, Martha K. (Inventor)

    2015-01-01

    Elongated microcapsules, such as elongated hydrophobic-core and hydrophilic-core microcapsules, may be formed by pulse stirring an emulsion or shearing an emulsion between two surfaces moving at different velocities. The elongated microcapsules may be dispersed in a coating formulation, such as paint.

  19. Seeds' physicochemical traits and mucilage protection against aluminum effect during germination and root elongation as important factors in a biofuel seed crop (Ricinus communis).

    PubMed

    Silva, Giovanni Eustáquio Alves; Ramos, Flávia Toledo; de Faria, Ana Paula; França, Marcel Giovanni Costa

    2014-10-01

    We determined the length, volume, dry biomass, and density in seeds of five castor bean cultivars and verified notable physicochemical trait differences. Seeds were then subjected to different toxic aluminum (Al) concentrations to evaluate germination, relative root elongation, and the role of root apices' rhizosphere mucilage layer. Seeds' physicochemical traits were associated with Al toxicity responses, and the absence of Al in cotyledons near to the embryo was revealed by Al-hematoxylin staining, indicating that Al did not induce significant germination reduction rates between cultivars. However, in the more sensitive cultivar, Al was found around the embryo, contributing to subsequent growth inhibition. After this, to investigate the role of mucilage in Al tolerance, an assay was conducted using NH4Cl to remove root mucilage before or after exposure to different Al concentrations. Sequentially, the roots were stained with hematoxylin and a quantitative analysis of staining intensity was obtained. These results revealed the significant contribution of the mucilage layer to Al toxicity responses in castor bean seedlings. Root growth elongation under Al toxicity confirmed the role of the mucilage layer, which jointly indicated the differential Al tolerance between cultivars and an efficient Al-exclusion mechanism in the tolerant cultivar.

  20. Cell division versus cell elongation: the control of radicle elongation during thermoinhibition of Tagetes minuta achenes.

    PubMed

    Taylor, Nicky J; Hills, Paul N; van Staden, Johannes

    2007-12-01

    Endogenous embryo factors, which act mainly in the radicle, prevent germination in Tagetes minuta at high temperatures. These factors act to prevent cell elongation, which is critical for radicle protrusion under optimal conditions. Once the radicle has emerged both cell elongation and cell division are required for post-germination growth. Germination can be induced at high temperatures by fusicoccin, which rapidly stimulates cell elongation. In addition, priming seeds at 25 degrees C on polyethylene glycol (PEG) 6000 and mannitol could also induce germination on water at 36 degrees C, indicating that priming prevents radicle protrusion at a point subsequent to the point of control in thermoinhibited achenes. Flow cytometry studies revealed that DNA synthesis occurs during thermoinhibition and the inhibition of DNA synthesis during this process inhibits subsequent germination on water under optimal conditions, suggesting a protective role for DNA synthesis in thermoinhibited achenes of T. minuta.

  1. A NusG paralogue from Mycobacterium tuberculosis, Rv0639, has evolved to interact with ribosomal protein S10 (Rv0700) but not to function as a transcription elongation-termination factor.

    PubMed

    Kalyani, B Sudha; Kunamneni, Radhika; Wal, Megha; Ranjan, Amitabh; Sen, Ranjan

    2015-01-01

    NusG, a well-conserved protein in all the three forms of life, is involved in transcription elongation and termination, as well as in the process of transcription-translation coupling. The existence of species-specific functional, as well as conformational, divergences in NusG makes it an attractive transcription factor to study, especially if it originates from a pathogen. Here, we report functional and conformational characterizations of the Mycobacterium tuberculosis (Mtb) protein Rv0639 that has been annotated as a homologue of Escherichia coli NusG. Rv0639 failed to complement the in vivo functions of E. coli NusG (Ec NusG) and did not exhibit any signature of a transcription elongation-termination factor. However, it retained the ability to bind to its cognate ribosomal protein S10 (Rv0700). Compared with Ec NusG, Rv0639 possesses unique conformational features characterized by altered secondary structures in the C-terminal domain (CTD), an unusually long and disordered linker region between the N-terminal domain (NTD) and CTD, and a folding of its NTD over its CTD. This unusual folded conformation could have imparted specialized functions to this protein, required to adapt the physiology of Mtb. We speculate that in the absence of a bona fide RfaH, a NusG paralogue that is involved in pathogenicity in E. coli, Rv0639 functions as an RfaH-like factor and is involved in pathogenicity using unidentified ops-like sequences in the Mtb genome. And hence, we reannotate Rv0639 as a paralogue of NusG, instead of a homologue.

  2. Morphological and Chemical Mechanisms of Elongated Mineral Particle Toxicities

    EPA Science Inventory

    Much of our understanding regarding the mechanisms for induction of disease following inhalation of respirable elongated mineral particles (REMPs) is based on studies involving the biological effects of asbestos fibers. The factors governing the disease potential of an exposure i...

  3. Functional interplay between PPM1G and the transcription elongation machinery

    PubMed Central

    Gudipaty, Swapna Aravind; D’Orso, Iván

    2016-01-01

    Transcription elongation is a critical regulatory step in the gene expression cycle. One key regulator of the switch between transcription initiation and elongation is the P-TEFb kinase, which phosphorylates RNA polymerase II (Pol II) and several negative elongation factors to relieve the elongation block at paused promoters to facilitate productive elongation. Here, we highlight recent findings signifying the role of the PPM1G/PP2Cγ phosphatase in activating and maintaining the active transcription elongation state by regulating the availability of P-TEFb and blocking its assembly into the catalytic inactive 7SK small nuclear ribonucleoprotein (snRNP) complex. PMID:27088130

  4. Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

    PubMed Central

    García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

    2015-01-01

    Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

  5. The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha is conserved among angiosperms.

    PubMed

    Curie, C; Liboz, T; Montané, M H; Rouan, D; Axelos, M; Lescure, B

    1992-04-01

    In Arabidopsis thaliana, the activation process of the A1 EF-1 alpha gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5' non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5' intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 alpha promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 alpha genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.

  6. Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

    PubMed

    Wilson, Gabriel J; Moulton, Christopher J; Garlick, Peter J; Anthony, Tracy G; Layman, Donald K

    2012-11-13

    Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced

  7. High elongation elastomers

    NASA Technical Reports Server (NTRS)

    Brady, V. L.; Reed, R.; Merwin, L.; Nissan, R.

    1994-01-01

    A new class of liquid curable elastomers with unusual strength and elasticity has been developed at the Naval Air Warfare Center Weapons Division, China Lake. Over the years, studies have been conducted on polymer structure and its influence on the mechanical properties of the ensuing composites. Different tools, including nuclear magnetic resonance, have been used. This paper presents a summary of the factors controlling the mechanical behavior of composites produced with the new liquid curable elastomers, including the effects of plasticizers. It also provides an overview of the nuclear magnetic resonance study on polymer structure, the composition and properties of some live and inert formulations produced at China Lake, and some possible peace-time applications for these new elastomeric materials.

  8. Mechanism of amyloid-β fibril elongation.

    PubMed

    Gurry, Thomas; Stultz, Collin M

    2014-11-11

    Amyloid-β is an intrinsically disordered protein that forms fibrils in the brains of patients with Alzheimer's disease. To explore factors that affect the process of fibril growth, we computed the free energy associated with disordered amyloid-β monomers being added to growing amyloid fibrils using extensive molecular dynamics simulations coupled with umbrella sampling. We find that the mechanisms of Aβ40 and Aβ42 fibril elongation have many features in common, including the formation of an obligate on-pathway β-hairpin intermediate that hydrogen bonds to the fibril core. In addition, our data lead to new hypotheses for how fibrils may serve as secondary nucleation sites that can catalyze the formation of soluble oligomers, a finding in agreement with recent experimental observations. These data provide a detailed mechanistic description of amyloid-β fibril elongation and a structural link between the disordered free monomer and the growth of amyloid fibrils and soluble oligomers.

  9. Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective.

    PubMed

    Brannan, Kris; Bentley, David L

    2012-01-01

    The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression. PMID:22567377

  10. Chromatin modification by the RNA Polymerase II elongation complex

    PubMed Central

    Tanny, Jason C.

    2014-01-01

    Transcription elongation by RNA polymerase II (RNAP II) involves the coordinated action of numerous regulatory factors. Among these are chromatin-modifying enzymes, which generate a stereotypic and conserved pattern of histone modifications along transcribed genes. This pattern implies a precise coordination between regulators of histone modification and the RNAP II elongation complex. Here I review the pathways and molecular events that regulate co-transcriptional histone modifications. Insight into these events will illuminate the assembly of functional RNAP II elongation complexes and how the chromatin landscape influences their composition and function. PMID:25494544

  11. Disruption and overexpression of auxin response factor 8 gene of Arabidopsis affect hypocotyl elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition.

    PubMed

    Tian, Chang-En; Muto, Hideki; Higuchi, Kanako; Matamura, Tomoyuki; Tatematsu, Kiyoshi; Koshiba, Tomokazu; Yamamoto, Kotaro T

    2004-11-01

    Auxin response factor (ARF) family genes play a central role in controlling sensitivity to the plant hormone auxin. We characterized the function of ARF8 in Arabidopsis by investigating a T-DNA insertion line (arf8-1) and overexpression lines (ARF8 OX) of ARF8. arf8-1 showed a long-hypocotyl phenotype in either white, blue, red or far-red light conditions, in contrast to ARF8 OX that displayed short hypocotyls in the light. Stronger and weaker apical dominance, and promotion and inhibition of lateral root formation were observed in arf8-1 and ARF8 OX respectively. Sensitivity to auxin was unaltered in arf8-1 hypocotyls with respect to growth inhibition caused by exogenously applied auxin and growth promotion induced by higher temperatures. ARF8 expression was observed constitutively in shoot and root apexes, and was induced in the light condition in hypocotyls. Free IAA contents were approximately 30% reduced in light-grown hypocotyls of ARF8 OX, but were similar between those of arf8-1 and wild type. Expression of the three GH3 genes was reduced in arf8-1 and increased in ARF8 OX, indicating that they are targets of ARF8 transcriptional control. Because the three GH3 proteins may be involved in the conjugation of IAA as suggested by Staswick et al. (2002), and because two of the three GH3 genes are auxin inducible, ARF8 may control the free IAA level in a negative feedback fashion by regulating GH3 gene expression. ARF family genes seem to control both auxin sensitivity and homeostasis in Arabidopsis.

  12. Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha

    SciTech Connect

    Whiteheart, S.W.; Shenbagamurthi, P.; Chen, L.; Cotter, R.J.; Hart, G.W. )

    1989-08-25

    Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with ({sup 3}H) ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major ({sup 3}H)ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release ({sup 3}H)ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues.

  13. Grain Size Dependence of Uniform Elongation in Single-Phase FCC/BCC Metals

    NASA Astrophysics Data System (ADS)

    Liu, Haiting; Shen, Yao; Ma, Jiawei; Zheng, Pengfei; Zhang, Lei

    2016-07-01

    We studied the dependence of uniform elongation on grain size in the range of submicron to millimeter for single-phase FCC/BCC metals by reviewing recent experimental results and applying crystal plasticity finite element method simulation. In the order of increasing grain size, uniform elongation can be divided into three stages, namely low elongation stage, nearly constant elongation stage, and decreased elongation with large scatters stage. Low elongation stage features a dramatic increase near the critical grain size at the end of the stage, which is primarily attributed to the emergence of dislocation cell size transition from ultrafine to mid-size grain. Other factors can be neglected due to their negligible influence on overall variation trend. In nearly constant elongation stage, uniform elongation remains unchanged at a high level in general. As grain size keeps growing, uniform elongation starts decreasing and becomes scattered upon a certain grain size, indicating the initiation of decreased elongation with large scatters stage. It is shown that the increase is not linear or smooth but rather sharp at the end of low elongation stage, leading to a wider range in nearly constant elongation stage. The grain size dependence of uniform elongation can serve as a guiding principle for designing small uniaxial tensile specimens for mechanical testing, where size effect matters in most cases.

  14. Grain Size Dependence of Uniform Elongation in Single-Phase FCC/BCC Metals

    NASA Astrophysics Data System (ADS)

    Liu, Haiting; Shen, Yao; Ma, Jiawei; Zheng, Pengfei; Zhang, Lei

    2016-09-01

    We studied the dependence of uniform elongation on grain size in the range of submicron to millimeter for single-phase FCC/BCC metals by reviewing recent experimental results and applying crystal plasticity finite element method simulation. In the order of increasing grain size, uniform elongation can be divided into three stages, namely low elongation stage, nearly constant elongation stage, and decreased elongation with large scatters stage. Low elongation stage features a dramatic increase near the critical grain size at the end of the stage, which is primarily attributed to the emergence of dislocation cell size transition from ultrafine to mid-size grain. Other factors can be neglected due to their negligible influence on overall variation trend. In nearly constant elongation stage, uniform elongation remains unchanged at a high level in general. As grain size keeps growing, uniform elongation starts decreasing and becomes scattered upon a certain grain size, indicating the initiation of decreased elongation with large scatters stage. It is shown that the increase is not linear or smooth but rather sharp at the end of low elongation stage, leading to a wider range in nearly constant elongation stage. The grain size dependence of uniform elongation can serve as a guiding principle for designing small uniaxial tensile specimens for mechanical testing, where size effect matters in most cases.

  15. Scattering from polymer networks under elongational strain

    NASA Astrophysics Data System (ADS)

    Svaneborg, C.; Grest, G. S.; Everaers, R.

    2005-12-01

    Molecular-dynamics simulations are used to sample the single-chain form factor of labelled sub-chains in model polymer networks under elongational strain. We observe very similar results for randomly cross-linked and for randomly end-linked networks with the same average strand length and see no indication of lozenge-like scattering patterns reported for some experimental systems. Our data analysis shows that a recent variant of the tube model quantitatively describes scattering in the Guinier regime as well as the macroscopic elastic properties. The observed failure of the theory outside the Guinier regime is shown to be due to non-Gaussian pair-distance distributions.

  16. Scattering from polymer networks under elongational strain.

    SciTech Connect

    Grest, Gary Stephen; Svaneborg, Carsten; Everaers, Ralf

    2005-06-01

    Molecular-dynamics simulations are used to sample the single-chain form factor of labelled sub-chains in model polymer networks under elongational strain. We observe very similar results for randomly cross-linked and for randomly end-linked networks with the same average strand length and see no indication of lozenge-like scattering patterns reported for some experimental systems. Our data analysis shows that a recent variant of the tube model quantitatively describes scattering in the Guinier regime as well as the macroscopic elastic properties. The observed failure of the theory outside the Guinier regime is shown to be due to non-Gaussian pair-distance distributions.

  17. What doesn’t kill them makes them stronger: an association between elongation factor 1-α overdominance in the sea star Pisaster ochraceus and “sea star wasting disease”

    PubMed Central

    Schiebelhut, Lauren M.

    2016-01-01

    In recent years, a massive mortality event has killed millions of sea stars, of many different species, along the Pacific coast of North America. This disease event, known as ‘sea star wasting disease’ (SSWD), is linked to viral infection. In one affected sea star (Pisaster ochraceus), previous work had identified that the elongation factor 1-α locus (EF1A) harbored an intronic insertion allele that is lethal when homozygous yet appears to be maintained at moderate frequency in populations through increased fitness for heterozygotes. The environmental conditions supporting this increased fitness are unknown, but overdominance is often associated with disease. Here, we evaluate populations of P. ochraceus to identify the relationship between SSWD and EF1A genotype. Our data suggest that there may be significantly decreased occurrence of SSWD in individuals that are heterozygous at this locus. These results suggest further studies are warranted to understand the functional relationship between diversity at EF1A and survival in P. ochraceus. PMID:27069810

  18. Direct Characterization of Transcription Elongation by RNA Polymerase I

    PubMed Central

    Ucuncuoglu, Suleyman; Engel, Krysta L.; Purohit, Prashant K.; Dunlap, David D.; Schneider, David A.

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  19. Direct Characterization of Transcription Elongation by RNA Polymerase I.

    PubMed

    Ucuncuoglu, Suleyman; Engel, Krysta L; Purohit, Prashant K; Dunlap, David D; Schneider, David A; Finzi, Laura

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  20. Amyloid-Like Fibril Elongation Follows Michaelis-Menten Kinetics

    PubMed Central

    Milto, Katazyna; Botyriute, Akvile; Smirnovas, Vytautas

    2013-01-01

    A number of proteins can aggregate into amyloid-like fibrils. It was noted that fibril elongation has similarities to an enzymatic reaction, where monomers or oligomers would play a role of substrate and nuclei/fibrils would play a role of enzyme. The question is how similar these processes really are. We obtained experimental data on insulin amyloid-like fibril elongation at the conditions where other processes which may impact kinetics of fibril formation are minor and fitted it using Michaelis-Menten equation. The correlation of the fit is very good and repeatable. It speaks in favour of enzyme-like model of fibril elongation. In addition, obtained and values at different conditions may help in better understanding influence of environmental factors on the process of fibril elongation. PMID:23874721

  1. Hack's Law: Sinuosity, convexity, elongation

    NASA Astrophysics Data System (ADS)

    Willemin, James H.

    2000-11-01

    Hack's law, an empirical, power law relationship between drainage basin area and the length of the main stream channel, has long been taken to imply that drainage basins become more elongate (relatively longer and narrower) with increasing basin size. A study of the geometry of 38 basins from three distinct geomorphic settings shows that this geometric interpretation of Hack's law is only occasionally true: Even though Hack's power law relationship holds between basin area and main channel length, these basins do not necessarily become more elongate with increasing size. Rather, Hack's law is an expression of a balance between changes in basin shape and changes in channel planform geometry. For the basins in this study, changes in channel sinuosity play the most important role in this balance; changes in basin shape are far less regular. Local conditions appear to determine the partitioning of importance between changes in basin shape and channel sinuosity.

  2. In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.

    PubMed

    Luker, Gary D; Luker, Kathryn E; Sharma, Vijay; Pica, Christina M; Dahlheimer, Julie L; Ocheskey, Joe A; Fahrner, Timothy J; Milbrandt, Jeffrey; Piwnica-Worms, David

    2002-01-01

    Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques. PMID:12920846

  3. The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment

    PubMed Central

    Loyola, Rodrigo; Herrera, Daniela; Mas, Abraham; Wong, Darren Chern Jan; Höll, Janine; Cavallini, Erika; Amato, Alessandra; Azuma, Akifumi; Ziegler, Tobias; Aquea, Felipe; Castellarin, Simone Diego; Bogs, Jochen; Tornielli, Giovanni Battista; Peña-Neira, Alvaro; Czemmel, Stefan; Alcalde, José Antonio; Matus, José Tomás; Arce-Johnson, Patricio

    2016-01-01

    Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera. We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures. PMID:27543604

  4. METHOD OF FORMING ELONGATED COMPACTS

    DOEpatents

    Larson, H.F.

    1959-05-01

    A powder compacting procedure and apparatus which produces elongated compacts of Be is described. The powdered metal is placed in a thin metal tube which is chemically compatible to lubricant, powder, atmosphere, and die material and will undergo a high degree of plastic deformation and have intermediate hardness. The tube is capped and placed in the die, and punches are applied to the ends. During the compacting stroke the powder seizes the tube and a thickening and shortening of the tube occurs. The tube is easily removed from the die, split, and peeled from the compact. (T.R.H.)

  5. Structure and Elongation of fine Ladies’ Hosiery

    NASA Astrophysics Data System (ADS)

    Lozo, M.; Vrljicak, Z.

    2016-07-01

    On a sock-knitting machine with diameter of cylindrical needle bed 100 mm (4e") that knitted with 400 needles, samples of fine women's hosiery were made from four PA filament yarns in counts 20 dtex f 20, 30 dtex f 34, 40 dtex f 40 and 60 dtex f 60. Each type of yarns was used to make hosiery samples with four loop sinking depths of unit values in a computer program 400, 550, 700 and 850. For all the samples, parameters of yarn structure were analyzed and elongation properties of knitted fabric were measured. During the elongation of knitted fabric, areas of knitted fabric elasticity, beginning of permanent deformation and elongation at break were measured. Elongation of knitted fabric in the wale direction, i.e. transverse hosiery elongation and elongation of knitted fabric in the course direction, or longitudinal direction of hosiery were measured. Yarn fineness and loop sinking depth significantly influence the elongation properties of hosiery.

  6. Th1 Stimulatory Proteins of Leishmania donovani: Comparative Cellular and Protective Responses of rTriose Phosphate Isomerase, rProtein Disulfide Isomerase and rElongation Factor-2 in Combination with rHSP70 against Visceral Leishmaniasis

    PubMed Central

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  7. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  8. A ratchet mechanism of transcription elongation and its control.

    PubMed

    Bar-Nahum, Gil; Epshtein, Vitaly; Ruckenstein, Andrei E; Rafikov, Ruslan; Mustaev, Arkady; Nudler, Evgeny

    2005-01-28

    RNA chain elongation is a highly processive and accurate process that is finely regulated by numerous intrinsic and extrinsic signals. Here we describe a general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs such as pauses, terminators, and elongation factors. We show that E.coli RNAP moves by a complex Brownian ratchet mechanism, which acts prior to phosphodiester bond formation. The incoming substrate and the flexible F bridge domain of the catalytic center serve as two separate ratchet devices that function in concert to drive forward translocation. The adjacent G loop domain controls F bridge motion, thus keeping the proper balance between productive and inactive states of the elongation complex. This balance is critical for cell viability since it determines the rate, processivity, and fidelity of transcription.

  9. Bilateral elongated mandibular coronoid process in an Anatolian skull

    PubMed Central

    Çorumlu, Ufuk; Demir, Mehmet Tevfik; Pirzirenli, Mennan Ece

    2016-01-01

    Elongation or hyperplasia of coronoid process of mandible is rare condition characterized by abnormal bone development which cause malocclusion and the limited mouth opening. In this study, in an Anatolian skull, a case of bilateral elongation of mandibular coronoid process was presented. Levandoski panographic analysis was performed on the panoramic radiographie to determine the hyperplasia of the coronoid process. The right condylar process was exactly hyperplastic. The measurements of Kr-Go/Cd-Go were 95.10 mm/79.03 mm on right side and 97.53 mm/87.80 mm on left side. The ratio of Kr-Go/Cd-Go on the right side was 1.20. Elongated coronoid process is one of the factors cause mandibular hypomobility, it as reported here might lead to limited mouth opening. The knowledge of this variation or abnormality can be useful for the radiologist and surgeons and prevent misdiagnosis. PMID:27722017

  10. Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication

    PubMed Central

    2013-01-01

    Background Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. Results We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. Conclusions Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication PMID:24165037

  11. Elongated Deposits in Southern Elysium Planitia, Mars

    NASA Astrophysics Data System (ADS)

    Nussbaumer, J. W.

    2012-03-01

    In the Elysium Planitia region, deposits have elongated elevations that resemble terrestrial drumlins or yardangs. Drumlins and drumlin clusters are glacial landforms that have been extensively studied. In contrast, Yardangs are formed by wind.

  12. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  13. Visualization of large elongated DNA molecules.

    PubMed

    Lee, Jinyong; Kim, Yongkyun; Lee, Seonghyun; Jo, Kyubong

    2015-09-01

    Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

  14. Telomere elongation chooses TERRA ALTernatives.

    PubMed

    Arora, Rajika; Azzalin, Claus M

    2015-01-01

    Alternative Lengthening of Telomeres (ALT) mechanisms allow telomerase-negative immortal cells to buffer replicative telomere shortening. ALT is naturally active in a number of human cancers and might be selected upon telomerase inactivation. ALT is thought to operate through homologous recombination (HR) occurring between telomeric repeats from independent chromosome ends. Indeed, suppression of a number of HR factors impairs ALT cell proliferation. Yet, how HR is initiated at ALT telomeres remains elusive. Mounting evidence suggests that the long noncoding telomeric RNA TERRA renders ALT telomeres recombinogenic by forming RNA:DNA hybrids with the telomeric C-rich strand. TERRA and telomeric hybrids act in concert with a number of other factors, including the RNA endoribonuclease RNaseH1 and the single stranded DNA binding protein RPA. The functional interaction network built upon these different players seems indispensable for ALT telomere maintenance, and digging into the molecular details of this previously unappreciated network might open the way to novel avenues for cancer treatments.

  15. Noise regulation and symmetry breaking during vertebrate body elongation

    NASA Astrophysics Data System (ADS)

    Emonet, Thierry; Das, Dipjyoti; Holley, Scott A.

    Elongation of the vertebrate body axis is driven by collective cell migration and cell proliferation at the posteriorly advancing embryonic tailbud. Within the Zebrafish tailbud an ordered stream of cells symmetrically bifurcates to form the left and right halves of the presomitic mesoderm. Maintaining bilateral symmetry during this process is critical to avoid catastrophic spine deformation. Using direct comparison between experimental data and a simple model of cell migration we identified the dynamic regulation of the noise in the direction of motion of individual cells as a critical factor in maintaining symmetric cell flow. Genetic perturbations that reduced noise led to body axis deformation whereas an increase in noise led to retarded elongation as predicted by our model.

  16. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans.

    PubMed

    Ahn, Jeong H; Rechsteiner, Andreas; Strome, Susan; Kelly, William G

    2016-08-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3' end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  17. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

    PubMed Central

    Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

    2016-01-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  18. Ethylene-promoted Elongation: an Adaptation to Submergence Stress

    PubMed Central

    Jackson, Michael B.

    2008-01-01

    Background A sizeable minority of taxa is successful in areas prone to submergence. Many such plants elongate with increased vigour when underwater. This helps to restore contact with the aerial environment by shortening the duration of inundation. Poorly adapted species are usually incapable of this underwater escape. Scope Evidence implicating ethylene as the principal factor initiating fast underwater elongation by leaves or stems is evaluated comprehensively along with its interactions with other hormones and gases. These interactions make up a sequence of events that link the perception of submergence to a prompt acceleration of extension. The review encompasses whole plant physiology, cell biology and molecular genetics. It includes assessments of how submergence threatens plant life and of the extent to which the submergence escape demonstrably improves the likelihood of survival. Conclusions Experimental testing over many years establishes ethylene-promoted underwater extension as one of the most convincing examples of hormone-mediated stress adaptation by plants. The research has utilized a wide range of species that includes numerous angiosperms, a fern and a liverwort. It has also benefited from detailed physiological and molecular studies of underwater elongation by rice (Oryza sativa) and the marsh dock (Rumex palustris). Despite complexities and interactions, the work reveals that the signal transduction pathway is initiated by the simple expediency of physical entrapment of ethylene within growing cells by a covering of water. PMID:17956854

  19. Reorientation of elongated particles at density interfaces.

    PubMed

    Doostmohammadi, A; Ardekani, A M

    2014-09-01

    Density interfaces in the water column are ubiquitously found in oceans and lakes. Interaction of settling particles with pycnoclines plays a pivotal function in nutrient transport between ocean layers and settling rates of marine particles. We perform direct numerical simulations of an elongated particle settling through a density interface and scrutinize the role of stratification on the settling dynamics. It is found that the presence of the density interface tends to turn the long axis of an elongated particle parallel to the settling direction, which is dramatically different from its counterpart in a homogeneous fluid. Although broadside-on settling of the elongated particle is enhanced upon approaching the interface, the long axis rotates toward the settling direction as the particle passes through the interface. We quantify turning couples due to stratification effects, which counteract the pressure-induced torques due to the fluid inertia. A similar behavior is observed for different initial orientations of the particle. It is shown that the reorientation of an elongated particle occurs in both sharp and linear density stratifications. PMID:25314535

  20. Interplay between DNA supercoiling and transcription elongation.

    PubMed

    Ma, Jie; Wang, Michelle

    2014-01-01

    Transcription-coupled DNA supercoiling has been shown to be an important regulator of transcription that is broadly present in the cell. Here we review experimental work which shows that RNA polymerase is a powerful torsional motor that can alter DNA topology and structure, and DNA supercoiling in turn directly affects transcription elongation.

  1. The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium.

    PubMed

    Pacheco-Villalobos, David; Díaz-Moreno, Sara M; van der Schuren, Alja; Tamaki, Takayuki; Kang, Yeon Hee; Gujas, Bojan; Novak, Ondrej; Jaspert, Nina; Li, Zhenni; Wolf, Sebastian; Oecking, Claudia; Ljung, Karin; Bulone, Vincent; Hardtke, Christian S

    2016-05-01

    The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots. PMID:27169463

  2. Abscisic Acid Regulates Root Elongation Through the Activities of Auxin and Ethylene in Arabidopsis thaliana

    PubMed Central

    Thole, Julie M.; Beisner, Erin R.; Liu, James; Venkova, Savina V.; Strader, Lucia C.

    2014-01-01

    Abscisic acid (ABA) regulates many aspects of plant growth and development, including inhibition of root elongation and seed germination. We performed an ABA resistance screen to identify factors required for ABA response in root elongation inhibition. We identified two classes of Arabidopsis thaliana AR mutants that displayed ABA-resistant root elongation: those that displayed resistance to ABA in both root elongation and seed germination and those that displayed resistance to ABA in root elongation but not in seed germination. We used PCR-based genotyping to identify a mutation in ABA INSENSITIVE2 (ABI2), positional information to identify mutations in AUXIN RESISTANT1 (AUX1) and ETHYLENE INSENSITIVE2 (EIN2), and whole genome sequencing to identify mutations in AUX1, AUXIN RESISTANT4 (AXR4), and ETHYLENE INSENSITIVE ROOT1/PIN-FORMED2 (EIR1/PIN2). Identification of auxin and ethylene response mutants among our isolates suggested that auxin and ethylene responsiveness were required for ABA inhibition of root elongation. To further our understanding of auxin/ethylene/ABA crosstalk, we examined ABA responsiveness of double mutants of ethylene overproducer1 (eto1) or ein2 combined with auxin-resistant mutants and found that auxin and ethylene likely operate in a linear pathway to affect ABA-responsive inhibition of root elongation, whereas these two hormones likely act independently to affect ABA-responsive inhibition of seed germination. PMID:24836325

  3. Abscisic acid regulates root elongation through the activities of auxin and ethylene in Arabidopsis thaliana.

    PubMed

    Thole, Julie M; Beisner, Erin R; Liu, James; Venkova, Savina V; Strader, Lucia C

    2014-05-15

    Abscisic acid (ABA) regulates many aspects of plant growth and development, including inhibition of root elongation and seed germination. We performed an ABA resistance screen to identify factors required for ABA response in root elongation inhibition. We identified two classes of Arabidopsis thaliana AR mutants that displayed ABA-resistant root elongation: those that displayed resistance to ABA in both root elongation and seed germination and those that displayed resistance to ABA in root elongation but not in seed germination. We used PCR-based genotyping to identify a mutation in ABA INSENSITIVE2 (ABI2), positional information to identify mutations in AUXIN RESISTANT1 (AUX1) and ETHYLENE INSENSITIVE2 (EIN2), and whole genome sequencing to identify mutations in AUX1, AUXIN RESISTANT4 (AXR4), and ETHYLENE INSENSITIVE ROOT1/PIN-FORMED2 (EIR1/PIN2). Identification of auxin and ethylene response mutants among our isolates suggested that auxin and ethylene responsiveness were required for ABA inhibition of root elongation. To further our understanding of auxin/ethylene/ABA crosstalk, we examined ABA responsiveness of double mutants of ethylene overproducer1 (eto1) or ein2 combined with auxin-resistant mutants and found that auxin and ethylene likely operate in a linear pathway to affect ABA-responsive inhibition of root elongation, whereas these two hormones likely act independently to affect ABA-responsive inhibition of seed germination.

  4. The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium[OPEN

    PubMed Central

    Pacheco-Villalobos, David; Tamaki, Takayuki; Gujas, Bojan; Jaspert, Nina; Oecking, Claudia; Bulone, Vincent; Hardtke, Christian S.

    2016-01-01

    The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots. PMID:27169463

  5. Tbx1 is Necessary for Palatal Elongation and Elevation

    PubMed Central

    Goudy, Steven; Law, Amy; Sanchez, Gabriela; Baldwin, H. Scott; Brown, Christopher

    2010-01-01

    The transcription factor TBX1 is a key mediator of developmental abnormalities associated with DiGeorge/Velocardiofacial Syndrome. Studies in mice have demonstrated that decreased dosage of Tbx1 results in defects in pharyngeal arch, cardiovascular, and craniofacial development. The role of Tbx1 in cardiac development has been intensely studied; however, its role in palatal development is poorly understood. By studying the Tbx1-/- mice we found defects during the critical points of palate elongation and elevation. The intrinsic palate defects in the Tbx1-/- mice were determined by measuring changes in palate shelf length, proliferation, apoptosis, expression of relevant growth factors, and in palate fusion assays. Tbx1-/- embryos exhibit cleft palate with failed palate elevation in 100% and abnormal palatal-oral fusions in 50%. In the Tbx1-/- mice the palate shelf length was reduced and tongue height was greater, demonstrating a physical impediment to palate elevation and apposition. In vitro palate fusion assays demonstrate that Tbx1-/- palate shelves are capable of fusion but a roller culture assay showed that the null palatal shelves were unable to elongate. Diminished hyaluronic acid production in the Tbx1-/- palate shelves may explain failed palate shelf elevation. In addition, cell proliferation and apoptosis were perturbed in Tbx1-/- palates. A sharp decrease of Fgf8 expression was detected in the Tbx1-/- palate shelves, suggesting that Fgf8 is dependent on Tbx1 in the palate. Fgf10 is also up-regulated in the Tbx1-/- palate shelves and tongue. These data demonstrate that Tbx1 is a critical transcription factor that guides palatal elongation and elevation and that Fgf8 expression in the palate is Tbx1-dependent. PMID:20214979

  6. Familial Occurrence of Enteric Muco-Submucosal Elongated Polyp

    PubMed Central

    Nakamura, Kenji; Okamoto, Takeshi; Imamura, Noriatsu; Ishii, Naoki; Fujita, Yoshiyuki

    2016-01-01

    We report 2 cases of enteric muco-submucosal elongated polyps (EMSEPs) that presented with gastrointestinal bleeding. The 2 patients are siblings. They both had a history of percutaneous coronary intervention for coronary artery disease and were on dual antiplatelet therapy. They underwent endoscopic resection of the polyps, which displayed identical endoscopic and histological features compatible with EMSEP. This is the first report of familial occurrence of EMSEP, suggesting possible genetic involvement. It is also important to note that the use of antiplatelet agents appears to be a predisposing factor for gastrointestinal bleeding from EMSEP. PMID:27807549

  7. Vertically stabilized elongated cross-section tokamak

    DOEpatents

    Sheffield, George V.

    1977-01-01

    This invention provides a vertically stabilized, non-circular (minor) cross-section, toroidal plasma column characterized by an external separatrix. To this end, a specific poloidal coil means is added outside a toroidal plasma column containing an endless plasma current in a tokamak to produce a rectangular cross-section plasma column along the equilibrium axis of the plasma column. By elongating the spacing between the poloidal coil means the plasma cross-section is vertically elongated, while maintaining vertical stability, efficiently to increase the poloidal flux in linear proportion to the plasma cross-section height to achieve a much greater plasma volume than could be achieved with the heretofore known round cross-section plasma columns. Also, vertical stability is enhanced over an elliptical cross-section plasma column, and poloidal magnetic divertors are achieved.

  8. Elongational viscosity of photo-oxidated LDPE

    SciTech Connect

    Rolón-Garrido, Víctor H. E-mail: manfred.wagner@tu-berlin.de; Wagner, Manfred H. E-mail: manfred.wagner@tu-berlin.de

    2014-05-15

    Sheets of low-density polyethylene (LDPE) were photo-oxidatively treated at room temperature, and subsequently characterized rheologically in the melt state by shear and uniaxial extensional experiments. For photo-oxidation, a xenon lamp was used to irradiate the samples for times between 1 day and 6 weeks. Linear-viscoelastic characterization was performed in a temperature range of 130 to 220°C to obtain the master curve at 170°C, the reference temperature at which the elongational viscosities were measured. Linear viscoelasticity is increasingly affected by increasing photo-oxidation due to crosslinking of LDPE, as corroborated by an increasing gel fraction as determined by a solvent extraction method. The elongational measurements reveal a strong enhancement of strain hardening until a saturation level is achieved. The elongational data are analyzed in the frame work of two constitutive equations, the rubber-like liquid and the molecular stress function models. Within the experimental window, timedeformation separability is confirmed for all samples, independent of the degree of photo-oxidation.

  9. Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq.

    PubMed

    Fuchs, Gilad; Voichek, Yoav; Rabani, Michal; Benjamin, Sima; Gilad, Shlomit; Amit, Ido; Oren, Moshe

    2015-04-01

    4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes ∼4 d to complete, with deep sequencing requiring an additional ∼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.

  10. Dimerization of elongator protein 1 is essential for Elongator complex assembly

    PubMed Central

    Xu, Huisha; Lin, Zhijie; Li, Fengzhi; Diao, Wentao; Dong, Chunming; Zhou, Hao; Xie, Xingqiao; Wang, Zheng; Shen, Yuequan; Long, Jiafu

    2015-01-01

    The evolutionarily conserved Elongator complex, which is composed of six subunits elongator protein 1 (Elp1 to -6), plays vital roles in gene regulation. The molecular hallmark of familial dysautonomia (FD) is the splicing mutation of Elp1 [also known as IκB kinase complex-associated protein (IKAP)] in the nervous system that is believed to be the primary cause of the devastating symptoms of this disease. Here, we demonstrate that disease-related mutations in Elp1 affect Elongator assembly, and we have determined the structure of the C-terminal portion of human Elp1 (Elp1-CT), which is sufficient for full-length Elp1 dimerization, as well as the structure of the cognate dimerization domain of yeast Elp1 (yElp1-DD). Our study reveals that the formation of the Elp1 dimer contributes to its stability in vitro and in vivo and is required for the assembly of both the human and yeast Elongator complexes. Functional studies suggest that Elp1 dimerization is essential for yeast viability. Collectively, our results identify the evolutionarily conserved dimerization domain of Elp1 and suggest that the pathological mechanisms underlying the onset and progression of Elp1 mutation-related disease may result from impaired Elongator activities. PMID:26261306

  11. Morphological and Chemical Mechanisms of Elongated Mineral Particle Toxicities

    PubMed Central

    Aust, Ann E.; Cook, Philip M.; Dodson, Ronald F.

    2011-01-01

    Much of our understanding regarding the mechanisms for induction of disease following inhalation of respirable elongated mineral particles (REMP) is based on studies involving the biological effects of asbestos fibers. The factors governing the disease potential of an exposure include duration and frequency of exposures; tissue-specific dose over time; impacts on dose persistence from in vivo REMP dissolution, comminution, and clearance; individual susceptibility; and the mineral type and surface characteristics. The mechanisms associated with asbestos particle toxicity involve two facets for each particle's contribution: (1) the physical features of the inhaled REMP, which include width, length, aspect ratio, and effective surface area available for cell contact; and (2) the surface chemical composition and reactivity of the individual fiber/elongated particle. Studies in cell-free systems and with cultured cells suggest an important way in which REMP from asbestos damage cellular molecules or influence cellular processes. This may involve an unfortunate combination of the ability of REMP to chemically generate potentially damaging reactive oxygen species, through surface iron, and the interaction of the unique surfaces with cell membranes to trigger membrane receptor activation. Together these events appear to lead to a cascade of cellular events, including the production of damaging reactive nitrogen species, which may contribute to the disease process. Thus, there is a need to be more cognizant of the potential impact that the total surface area of REMP contributes to the generation of events resulting in pathological changes in biological systems. The information presented has applicability to inhaled dusts, in general, and specifically to respirable elongated mineral particles. PMID:21534085

  12. Fruiting Branch K+ Level Affects Cotton Fiber Elongation Through Osmoregulation

    PubMed Central

    Yang, Jiashuo; Hu, Wei; Zhao, Wenqing; Chen, Binglin; Wang, Youhua; Zhou, Zhiguo; Meng, Yali

    2016-01-01

    Potassium (K) deficiency in cotton plants results in reduced fiber length. As one of the primary osmotica, K+ contributes to an increase in cell turgor pressure during fiber elongation. Therefore, it is hypothesized that fiber length is affected by K deficiency through an osmotic pathway, so in 2012 and 2013, an experiment was conducted to test this hypothesis by imposing three potassium supply regimes (0, 125, 250 kg K ha-1) on a low-K-sensitive cultivar, Siza 3, and a low-K-tolerant cultivar, Simian 3. We found that fibers were longer in the later season bolls than in the earlier ones in cotton plants grown under normal growth conditions, but later season bolls showed a greater sensitivity to low-K stress, especially the low-K sensitive genotype. We also found that the maximum velocity of fibre elongation (Vmax) is the parameter that best reflects the change in fiber elongation under K deficiency. This parameter mostly depends on cell turgor, so the content of the osmotically active solutes was analyzed accordingly. Statistical analysis showed that K+ was the major osmotic factor affecting fiber length, and malate was likely facilitating K+ accumulation into fibers, which enabled the low-K-tolerant genotype to cope with low-K stress. Moreover, the low-K-tolerant genotype tended to have greater K+ absorptive capacities in the upper fruiting branches. Based on our findings, we suggest a fertilization scheme for Gossypium hirsutum that adds extra potash fertilizer or distributes it during the development of late season bolls to mitigate K deficiency in the second half of the growth season and to enhance fiber length in late season bolls. PMID:26834777

  13. Fruiting Branch K(+) Level Affects Cotton Fiber Elongation Through Osmoregulation.

    PubMed

    Yang, Jiashuo; Hu, Wei; Zhao, Wenqing; Chen, Binglin; Wang, Youhua; Zhou, Zhiguo; Meng, Yali

    2016-01-01

    Potassium (K) deficiency in cotton plants results in reduced fiber length. As one of the primary osmotica, K(+) contributes to an increase in cell turgor pressure during fiber elongation. Therefore, it is hypothesized that fiber length is affected by K deficiency through an osmotic pathway, so in 2012 and 2013, an experiment was conducted to test this hypothesis by imposing three potassium supply regimes (0, 125, 250 kg K ha(-1)) on a low-K-sensitive cultivar, Siza 3, and a low-K-tolerant cultivar, Simian 3. We found that fibers were longer in the later season bolls than in the earlier ones in cotton plants grown under normal growth conditions, but later season bolls showed a greater sensitivity to low-K stress, especially the low-K sensitive genotype. We also found that the maximum velocity of fibre elongation (V max) is the parameter that best reflects the change in fiber elongation under K deficiency. This parameter mostly depends on cell turgor, so the content of the osmotically active solutes was analyzed accordingly. Statistical analysis showed that K(+) was the major osmotic factor affecting fiber length, and malate was likely facilitating K(+) accumulation into fibers, which enabled the low-K-tolerant genotype to cope with low-K stress. Moreover, the low-K-tolerant genotype tended to have greater K(+) absorptive capacities in the upper fruiting branches. Based on our findings, we suggest a fertilization scheme for Gossypium hirsutum that adds extra potash fertilizer or distributes it during the development of late season bolls to mitigate K deficiency in the second half of the growth season and to enhance fiber length in late season bolls. PMID:26834777

  14. Nonlinear deformations of microcapsules in elongation flow

    NASA Astrophysics Data System (ADS)

    Deschamps, Julien; de Loubens, Clément; Boedec, Gwenn; Georgelin, Marc; Leonetti, Marc; Soft Matter; Biophysics Group Team

    2014-11-01

    Soft microcapsules are drops bounded by a thin elastic shell made of cross-linked proteins. They have numerous applications for drug delivery in bioengineering, pharmaceutics and medicine, where their mechanical stability and their dynamics under flow are crucial. They can also be used as red blood cells models. Here, we investigate the mechanical behaviour of microcapsules made of albumine in strong elongational flow, up to a stretching of 180% just before breaking. The set-up allows us to visualize the deformed shape in the two perpendicular main fields of view, to manage high capillary number and to manipulate soft microcapsules. The steady-state shape of a capsule in the planar elongational flow is non-axisymmetric. In each cross section, the shape is an ellipse but with different small axis which vary in opposite sense with the stretching. Whatever the degree of cross-linking and the size of the capsules, the deformations followed the same master-curve. Comparisons between numerical predictions and experimental results permit to conclude unambiguously that the more properly strain-energy model of membrane is the generalized Hooke model.

  15. Trade studies of plasma elongation for next-step tokamaks

    SciTech Connect

    Galambos, J.D.; Strickler, D.J.; Peng, Y.K.M.; Reid, R.L.

    1988-09-01

    The effect of elongation on minimum-cost devices is investigated for elongations ranging from 2 to 3. The analysis, carried out with the TETRA tokamak systems code, includes the effects of elongation on both physics (plasma beta limit) and engineering (poloidal field coil currents) issues. When ignition is required, the minimum cost occurs for elongations from 2.3 to 2.9, depending on the plasma energy confinement scaling used. Scalings that include favorable plasma current dependence and/or degradation with fusion power tend to have minimum cost at higher elongation (2.5-2.9); scalings that depend primarily on size result in lower elongation (/approximately/2.3) for minimum cost. For design concepts that include steady-state current-driven operation, minimum cost occurs at an elongation of 2.3. 12 refs., 13 figs.

  16. Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

    PubMed Central

    Yuzenkova, Yulia; Gamba, Pamela; Herber, Martijn; Attaiech, Laetitia; Shafeeq, Sulman; Kuipers, Oscar P.; Klumpp, Stefan; Zenkin, Nikolay; Veening, Jan-Willem

    2014-01-01

    Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes. PMID:25190458

  17. Genetic evidence supports a role for the yeast CCR4-NOT complex in transcriptional elongation.

    PubMed Central

    Denis, C L; Chiang, Y C; Cui, Y; Chen, J

    2001-01-01

    The CCR4-NOT complex is involved in the regulation of gene expression both positively and negatively. The repressive effects of the complex appear to result in part from restricting TBP access to noncanonical TATAA binding sites presumably through interaction with multiple TAF proteins. We provide here genetic evidence that the CCR4-NOT complex also plays a role in transcriptional elongation. First, defects in CCR4-NOT components as well as overexpression of the NOT4 gene elicited 6-azauracil (6AU) and mycophenolic acid sensitivities, hallmarks of transcriptional elongation defects. A number of other transcription initiation factors known to interact with the CCR4-NOT complex did not elicit these phenotypes nor did defects in factors that reduced mRNA degradation and hence the recycling of NTPs. Second, deletion of ccr4 resulted in severe synthetic effects with mutations or deletions in the known elongation factors RPB2, TFIIS, and SPT16. Third, the ccr4 deletion displayed allele-specific interactions with rpb1 alleles that are thought to be important in the control of elongation. Finally, we found that a ccr4 deletion as well as overexpression of the NOT1 gene specifically suppressed the cold-sensitive phenotype associated with the spt5-242 allele. The only other known suppressors of this spt5-242 allele are factors involved in slowing transcriptional elongation. These genetic results are consistent with the model that the CCR4-NOT complex, in addition to its known effects on initiation, plays a role in aiding the elongation process. PMID:11404327

  18. Potential flow about elongated bodies of revolution

    NASA Technical Reports Server (NTRS)

    Kaplan, Carl

    1936-01-01

    This report presents a method of solving the problem of axial and transverse potential flows around arbitrary elongated bodies of revolution. The solutions of Laplace's equation for the velocity potentials of the axial and transverse flows, the system of coordinates being an elliptic one in a meridian plane, are given. The theory is applied to a body of revolution obtained from a symmetrical Joukowsky profile, a shape resembling an airship hull. The pressure distribution and the transverse-force distribution are calculated and serve as examples of the procedure to be followed in the case of an actual airship. A section on the determination of inertia coefficients is also included in which the validity of some earlier work is questioned.

  19. Low temperature viscosity in elongated ferrofluids

    NASA Astrophysics Data System (ADS)

    Alarcón, T.; Pérez-Madrid, A.; Rubí, J. M.

    1997-12-01

    We have studied the relaxation and transport properties of a ferrofluid in an elongational flow. These properties are influenced by the bistable nature of the potential energy. Bistability comes from the irrotational character of the flow together with the symmetry of the dipoles. Additionally, the presence of a constant magnetic field destroys the symmetry of the potential energy magnetizing the system. We have shown that at a moderate temperature, compared to the height of the energy barrier, the viscosity decreases with respect to the value it would have if the potential were stable. This phenomenon is known as the "negative viscosity" effect. Thermal motion induces jumps of the magnetic moment between the two stable states of the system leading to the aforementioned lowered dissipation effect.

  20. Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation

    PubMed Central

    Petralia, Ronald S.; Ott, Carolyn; Wang, Ya-Xian; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

    2015-01-01

    The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits. PMID:26658865

  1. Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate.

    PubMed

    García, Alicia; Collin, Alejandro; Calvo, Olga

    2012-11-01

    The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3'-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5-Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5-Rpb1 complex levels and consequently transcription elongation rate.

  2. Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate

    PubMed Central

    García, Alicia; Collin, Alejandro; Calvo, Olga

    2012-01-01

    The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3′-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5–Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5–Rpb1 complex levels and consequently transcription elongation rate. PMID:22973055

  3. Role of lipids on elongation of the preimplantation conceptus in ruminants.

    PubMed

    Ribeiro, Eduardo S; Santos, José E P; Thatcher, William W

    2016-10-01

    Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants.

  4. Role of lipids on elongation of the preimplantation conceptus in ruminants.

    PubMed

    Ribeiro, Eduardo S; Santos, José E P; Thatcher, William W

    2016-10-01

    Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants. PMID:27335133

  5. Conditions for bubble elongation in cold ice-sheet ice

    USGS Publications Warehouse

    Alley, R.B.; Fitzpatrick, J.J.

    1999-01-01

    Highly elongated bubbles are sometimes observed in ice-sheet ice. Elongation is favored by rapid ice deformation, and opposed by diffusive processes. We use simple models to show that vapor transport dominates diffusion except possibly very close to the melting point, and that latent-heat effects are insignificant. Elongation is favored by larger bubbles at pore close-off, but is nearly independent of bubble compression below close-off. The simple presence of highly elongated bubbles indicates only that a critical ice-strain rate has been exceeded for significant time, and provides no information on possible disruption of stratigraphic continuity by ice deformation.

  6. Venus - A Large Elongated Caldera 'Sacajawea Patera

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This Magellan image reveals Sacajawea Patera, a large, elongate caldera located in Western Ishtar Terra on the smooth plateau of Lakshmi Planum. The image is centered at 64.5 degrees North latitude and 337 degrees East longitude. It is approximately 420 kilometers (252 miles) wide at the base. Sacajawea is a depression approximately 1-2 kilometers (0.6-1.2 miles) deep and 120 x 215 kilometers (74 x 133 miles) in diameter; it is elongate in a southwest-northeast direction. The depression is bounded by a zone of circumferential curvilinear structures interpreted to be graben and fault scarps. These structures are spaced 0.5-4 kilometers (0.3-2.5 miles) apart, are 0.6-4.0 kilometers (0.4-2.5 miles) in width and up to 100 kilometers (62 miles) in length. Extending up to approximately 140 kilometers (87 miles) in length from the southeast of the patera is a system of linear structures thought to represent a flanking rift zone along which the lateral injection and eruption of magma may have occurred. A shield edifice 12 kilometers (7 miles) in diameter with a prominent central pit lies along the trend of one of these features. The impact crater Zlata, approximately 6 kilometers (4 miles) in diameter is located within the zone of graben to the northwest of the patera. Few flow features are observed in association with Sacajawea, possibly due to age and state of degradation of the flows. Mottled bright deposits 4-20 kilometers (2.5-12 miles) in width are located near the periphery and in the center of the patera floor within local topographic lows. Diffuse patches of dark material approximately 40 kilometers (25 miles) in width are observed southwest of the patera, superposed on portions of the surrounding graben. The formation of Sacajawea is thought to be related to the drainage and collapse of a large magma chamber. Gravitational relaxation may have caused the resultant caldera to sag, producing the numerous faults and graben that circumscribe the patera. Regions of

  7. Emerging brain morphologies from axonal elongation

    PubMed Central

    Holland, Maria A.; Miller, Kyle E.; Kuhl, Ellen

    2015-01-01

    Understanding the characteristic morphology of our brain remains a challenging, yet important task in human evolution, developmental biology, and neurosciences. Mathematical modeling shapes our understanding of cortical folding and provides functional relations between cortical wavelength, thickness, and stiffness. Yet, current mathematical models are phenomenologically isotropic and typically predict non-physiological, periodic folding patterns. Here we establish a mechanistic model for cortical folding, in which macroscopic changes in white matter volume are a natural consequence of microscopic axonal growth. To calibrate our model, we consult axon elongation experiments in chick sensory neurons. We demonstrate that a single parameter, the axonal growth rate, explains a wide variety of in vitro conditions including immediate axonal thinning and gradual thickness restoration. We embed our axonal growth model into a continuum model for brain development using axonal orientation distributions motivated by diffusion spectrum imaging. Our simulations suggest that white matter anisotropy - as an emergent property from directional axonal growth - intrinsically induces symmetry breaking, and predicts more physiological, less regular morphologies with regionally varying gyral wavelengths and sulcal depths. Mechanistic modeling of brain development could establish valuable relationships between brain connectivity, brain anatomy, and brain function. PMID:25824370

  8. Glycoproteome of Elongating Cotton Fiber Cells*

    PubMed Central

    Kumar, Saravanan; Kumar, Krishan; Pandey, Pankaj; Rajamani, Vijayalakshmi; Padmalatha, Kethireddy Venkata; Dhandapani, Gurusamy; Kanakachari, Mogilicherla; Leelavathi, Sadhu; Kumar, Polumetla Ananda; Reddy, Vanga Siva

    2013-01-01

    Cotton ovule epidermal cell differentiation into long fibers primarily depends on wall-oriented processes such as loosening, elongation, remodeling, and maturation. Such processes are governed by cell wall bound structural proteins and interacting carbohydrate active enzymes. Glycosylation plays a major role in the structural, functional, and localization aspects of the cell wall and extracellular destined proteins. Elucidating the glycoproteome of fiber cells would reflect its wall composition as well as compartmental requirement, which must be system specific. Following complementary proteomic approaches, we have identified 334 unique proteins comprising structural and regulatory families. Glycopeptide-based enrichment followed by deglycosylation with PNGase F and A revealed 92 unique peptides containing 106 formerly N-linked glycosylated sites from 67 unique proteins. Our results showed that structural proteins like arabinogalactans and carbohydrate active enzymes were relatively more abundant and showed stage- and isoform-specific expression patterns in the differentiating fiber cell. Furthermore, our data also revealed the presence of heterogeneous and novel forms of structural and regulatory glycoproteins. Comparative analysis with other plant glycoproteomes highlighted the unique composition of the fiber glycoproteome. The present study provides the first insight into the identity, abundance, diversity, and composition of the glycoproteome within single celled cotton fibers. The elucidated composition also indirectly provides clues about unicellular compartmental requirements underlying single cell differentiation. PMID:24019148

  9. Nonlocal order in elongated dipolar gases

    NASA Astrophysics Data System (ADS)

    Ruhman, J.; Dalla Torre, E. G.; Huber, S. D.; Altman, E.

    2012-03-01

    Dipolar particles in an elongated trap are expected to undergo a quantum phase transition from a linear to a zigzag structure with decreasing transverse confinement. We derive the low-energy effective theory of the transition showing that in the presence of quantum fluctuations the zigzag phase can be characterized by a long-ranged string order, while the local Ising correlations decay as a power law. This is also confirmed using density matrix renormalization group calculations on a microscopic model. The nonlocal order in the bulk gives rise to zero energy states localized at the interface between the ordered and disordered phases. Such an interface naturally arises when the particles are subject to a weak harmonic confinement along the tube axis. We compute the signature of the edge states in the single-particle tunneling spectra pointing to differences between a system with bosonic versus fermionic particles. Finally we assess the magnitude of the relevant quantum fluctuations in realistic systems of dipolar particles, including ultracold polar molecules as well as alkali atoms weakly dressed by a Rydberg excitation.

  10. Genome-Wide and Experimental Resolution of Relative Translation Elongation Speed at Individual Gene Level in Human Cells

    PubMed Central

    Gu, Wei; Cui, Yizhi; Zhong, Jiayong; Jin, Jingjie; He, Qing-Yu; Wang, Tong; Zhang, Gong

    2016-01-01

    In the process of translation, ribosomes first assemble on mRNAs (translation initiation) and then translate along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By integrating three types of RNA-seq methods, we experimentally and computationally resolved elongation speed, with our proposed elongation velocity index (EVI), a relative measure at individual gene level and under physiological condition in human cells. We successfully distinguished slow-translating genes from the background translatome. We demonstrated that low-EVI genes encoded more stable proteins. We further identified cell-specific slow-translating codons, which might serve as a causal factor of elongation deceleration. As an example for the biological relevance, we showed that the relatively slow-translating genes tended to be associated with the maintenance of malignant phenotypes per pathway analyses. In conclusion, EVI opens a new view to understand why human cells tend to avoid simultaneously speeding up translation initiation and decelerating elongation, and the possible cancer relevance of translating low-EVI genes to gain better protein quality. PMID:26926465

  11. RNAP II processivity is a limiting step for HIV-1 transcription independent of orientation to and activity of endogenous neighboring promoters.

    PubMed

    Kaczmarek Michaels, Katarzyna; Wolschendorf, Frank; Schiralli Lester, Gillian M; Natarajan, Malini; Kutsch, Olaf; Henderson, Andrew J

    2015-12-01

    Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency.

  12. Positive grid corrosion elongation analysis using CAE with corrosion deformation transformed into thermal phenomenon

    NASA Astrophysics Data System (ADS)

    Mukaitani, Ichiroh; Hayashi, Koji; Shimoura, Ichiro; Takemasa, Arihiko; Takahashi, Isamu; Tsubakino, Harushige

    Valve-regulated lead-acid (VRLA) batteries have been commercially available for more than 20 years and have been enthusiastically embraced by users of uninterruptible power supplies (UPS) because of the anticipated reduction in installation and operating costs, smaller footprint and fewer environmental concerns. In Japan, communication networks are demanding reduced costs and longer life from their batteries. Among the factors limiting the life of VRLA batteries, the corrosion of positive grid material has been proven to cause elongation of the plates, loss of electrical contact and shorter lifetime. The content of Sn is also a key factor and addition of Sn in the grid alloy results in better performance in creep resistance, tensile strength and corrosion resistance [R. David Prenagaman, The Battery Man, vol. 39, September 1997, p. 16. I. Mukaitani, T. Sakamoto, T. Kikuoka, Y. Yamaguchi, H. Tsubakino, Proceedings of the 40th Battery Symposium in Japan, 1999, p. 99]. A key point is what the ratio of Sn to Ca should be, since too much Sn may lead to even worse elongation of the plates [I. Mukaitani, T. Sakamoto, T. Kikuoka, Y. Yamaguchi, H. Tsubakino, Proceedings of the 40th Battery Symposium in Japan, 1999, p. 99]. We have determined that microstructure control with a composition of lead-calcium-tin (Pb-Ca-Sn) alloy is optimal for better performance of the plates [I. Mukaitani, T. Sakamoto, T. Kikuoka, Y. Yamaguchi, H. Tsubakino, Proceedings of the 40th Battery Symposium in Japan, 1999, p. 99]. We developed a "simulation of current collector corrosion elongation" which is a technique of estimating corrosion elongation from the current collector design [I. Mukaitani, K. Hayashi, I. Shimoura, H. Takabayashi, M. Terada, A. Takemasa, I. Takahashi, K. Okamoto, Proceedings of the 44th Battery Symposium in Japan, 2003, p. 652]. Corrosion elongation occurs as the corrosion material layer grows out of the current collector metal. We resolved this problem using generally CAD

  13. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    NASA Technical Reports Server (NTRS)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  14. Blastocyst Elongation, Trophoblastic Differentiation and Embryonic Pattern Formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular basis behind elongation and concomitant gastrulation in ungulates that occurs during pre-implantation is still poorly understood. In-depth transcriptome analysis of the elongating porcine conceptus at specific stages has demonstrated that protein synthesis, protein trafficking, cell g...

  15. Conceptus elongation in cattle: genes, models and questions.

    PubMed

    Hue, Isabelle; Degrelle, Séverine Aude; Turenne, Nicolas

    2012-09-01

    In ruminants, more than 30% of the embryonic loss observed after artificial insemination has an early origin that is coincident with the marked elongation of the conceptus that occurs before implantation. During this developmental phase, physiological interactions are established between the conceptus and the uterus which are essential for the establishment of pregnancy and the elongation process. Our molecular knowledge of elongating conceptuses in cattle has long been focused on its analysis in view of its interactions with the uterus with the elongating stages being defined, like the uterus stages, by days post insemination or conception. The gene clusters reported so far indicate important pathways, some being shared by the non-elongating conceptuses of other mammals. However, to identify the key components of the elongation process - that could be specific to ungulates - new models are needed. Somatic nuclear transfer could be one of them as it provides complementary insights on differentiation beyond the blastocyst stage. Nonetheless, other models are necessary to convert gene lists or networks in elongating phenotypes. This review partly summarizes information on these topics, but data on the impact of the uterus on the elongation process or on the differentiation of the embryonic tissues are reviewed elsewhere. PMID:22921267

  16. The Effects of Microgravity on Seated Height (Spinal Elongation)

    NASA Technical Reports Server (NTRS)

    Young, K. S.; Rajulu, S.

    2011-01-01

    ABSTRACT Many physiological factors, such as spinal elongation, fluid shifts, bone atrophy, and muscle loss, occur during an exposure to a microgravity environment. Spinal elongation is just one of the factors that can also affect the safety and performance of a crewmember while in space. Spinal elongation occurs due to the lack of gravity/compression on the spinal column. This allows for the straightening of the natural spinal curve. There is a possible fluid shift in the inter-vertebral disks that may also result in changes in height. This study aims at collecting the overall change in seated height for crewmembers exposed to a microgravity environment. During previous Programs, Apollo-Soyuz Test Project (ASTP) and Skylab, spinal elongation data was collected from a small number of subjects in a standing posture but were limited in scope. Data from these studies indicated a quick increase in stature during the first few days of weightlessness, after which stature growth reached a plateau resulting in up to a 3% increase of the original measurement [1-5]. However, this data was collected only for crewmembers in standing posture and not in a seated posture. Seated height may have a different effect than standing height due to a change in posture as well as due to a compounded effect of wearing restraints and a potential compression of the gluteal area. Seated height was deemed as a critical measurement in the design of the Constellation Program s (CxP) Crew Exploration Vehicle (CEV), called Orion which is now the point-of-departure vehicle for the Multi-Purpose Crew Vehicle (MPCV) Program; therefore a better understanding of the effects of microgravity on seated height is necessary. Potential changes in seated height that may not have impacted crew accommodation in previous Programs will have significant effects on crew accommodation due to the layout of seats in the Orion.. The current and existing configuration is such that the four crewmembers are stacked two by

  17. DksA Guards Elongating RNA Polymerase Against Ribosome-Stalling-Induced Arrest

    PubMed Central

    Zhang, Yan; Mooney, Rachel A.; Grass, Jeffrey A.; Sivaramakrishnan, Priya; Herman, Christophe; Landick, Robert; Wang, Jue D.

    2014-01-01

    Summary In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. PMID:24606919

  18. Formation of elongated galaxies with low masses at high redshift

    NASA Astrophysics Data System (ADS)

    Ceverino, Daniel; Primack, Joel; Dekel, Avishai

    2015-10-01

    We report the identification of elongated (triaxial or prolate) galaxies in cosmological simulations at z ≃ 2. These are preferentially low-mass galaxies (M* ≤ 109.5 M⊙), residing in dark matter (DM) haloes with strongly elongated inner parts, a common feature of high-redshift DM haloes in the Λ cold dark matter cosmology. Feedback slows formation of stars at the centres of these haloes, so that a dominant and prolate DM distribution gives rise to galaxies elongated along the DM major axis. As galaxies grow in stellar mass, stars dominate the total mass within the galaxy half-mass radius, making stars and DM rounder and more oblate. A large population of elongated galaxies produces a very asymmetric distribution of projected axis ratios, as observed in high-z galaxy surveys. This indicates that the majority of the galaxies at high redshifts are not discs or spheroids but rather galaxies with elongated morphologies.

  19. Sequence-dependent elongation dynamics on macrolide-bound ribosomes.

    PubMed

    Johansson, Magnus; Chen, Jin; Tsai, Albert; Kornberg, Guy; Puglisi, Joseph D

    2014-06-12

    The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

  20. Patterns of cell elongation in the determination of the final shape in galls of Baccharopelma dracunculifoliae (Psyllidae) on Baccharis dracunculifolia DC (Asteraceae).

    PubMed

    Magalhães, Thiago Alves; de Oliveira, Denis Coelho; Suzuki, Aline Yasko Marinho; Isaias, Rosy Mary dos Santos

    2014-07-01

    Cell redifferentiation, division, and elongation are recurrent processes, which occur during gall development, and are dependent on the cellulose microfibrils reorientation. We hypothesized that changes in the microfibrils orientation from non-galled tissues to galled ones occur and determine the final gall shape. This determination is caused by a new tissue zonation, its hyperplasia, and relative cell hypertrophy. The impact of the insect's activity on these patterns of cell development was herein tested in Baccharopelma dracunculifoliae-Baccharis dracunculifolia system. In this system, the microfibrils are oriented perpendicularly to the longest cell axis in elongated cells and randomly in isodiametric ones, either in non-galled or in galled tissues. The isodiametric cells of the abaxial epidermis in non-galled tissues divided and elongated periclinally, forming the outer gall epidermis. The anticlinally elongated cells of the abaxial palisade layer and the isodiametric cells of the spongy parenchyma originated the gall outer cortex with hypertrophied and periclinally elongated cells. The anticlinally elongated cells of the adaxial palisade layer originated the inner cortex with hypertrophied and periclinally elongated cells in young and mature galls and isodiametric cells in senescent galls. The isodiametric cells of the adaxial epidermis elongated periclinally in the inner gall epidermis. The current investigation demonstrates the role of cellulose microfibril reorientation for gall development. Once many factors other than this reorientation act on gall development, it should be interesting to check the possible relationship of the new cell elongation patterns with the pectic composition of the cell walls.

  1. [The roles of microtubule in internodal cell elongation (Nitellopsis obtusa)].

    PubMed

    Yu, Rong; Yuan, Ming; Zhu, Guo Li; Wang, Xue Chen

    2004-04-01

    The relationship between cell elongation and microtubules (MTs) was investigated in characean internodal cells (Nitellops obtusa). First, we examined the immunofluorescent localization of MTs in different living stages under confocal laser scanning microscope. In young, rapidly elongating cells, MTs were predominantly transverse to the long axis of the cell. As the relative growth rate fell, transverse MTs gradually decreased, and in non-growing cells, longitudinally oriented cortical MTs became most pronounced. Moreover, cells in different living stages responded to the treatment of oryzalin (microtubule-disrupting agent) differently, young active internodal cells seemed to be more sensitive. After 40 min incubation of 10 micromol/L oryzalin, nearly all cortical MTs in the elongating cells depolymerized. However, in the old, non-growing cells, some MT fragments still remained after 3 h treatment of oryzalin. Second, we measured the cell growth rates with and without the treatment of oryzalin. In young growing cells treated with 10 micromol/L oryzalin, the elongation rates were inhibited obviously. When the oryzalin was removed, the elongation rates could be recovered to some extent. Interestingly, a time-gap existed between microtubule disassembly (40 min) and cessation of cell elongation (100 min). Our data confirmed the evidence that MTs are involved in cell elongation.

  2. Elastocaloric effect dependence on pre-elongation in natural rubber

    NASA Astrophysics Data System (ADS)

    Xie, Zhongjian; Sebald, Gael; Guyomar, Daniel

    2015-08-01

    In the context of solid-state-cooling, the elastocaloric effect offers a very large controlled entropy change based in low-cost polymers, especially natural rubber which is environmentally friendly. However, large elastocaloric activity requires large elongation (>5), which makes this material impractical for cooling systems due to the large change in sample's area. By performing a pre-elongation, area change is limited, and β = - ∂ γ / ∂ λ (where γ is the specific entropy and λ is the elongation) is larger. The highest β value is obtained when pre-elongation is right before (at the "eve") the onset of the strain-induced crystallization, which is also interpreted in the view of molecular conformation. Experimental results obtained on a natural rubber sample showed an adiabatic temperature change of 4.3 °C for pre-elongation of 4 with further elongation of 4 (true strain change of 69%). Furthermore, the entropy exhibits a quasi-linear dependence on elongation, and the β value is found to be 6400 J K-1 m-3.

  3. Association between regulator of telomere elongation helicase 1 polymorphism and susceptibility to glioma

    PubMed Central

    Pei, Shujun; Zhao, Feng; Liu, Junle; Fu, Qiang; Shang, Peizhong

    2015-01-01

    Background: Glioma is the most devastating type of malignant brain tumors in adults. Genetic factors play important roles in the pathogenesis of glioma. In recent years, some studies found that there were significant association between regulator of telomere elongation helicase 1 rs6010620 polymorphism and glioma susceptibility, however, the results were controversial. The aim of this study was to obtain a more exact estimation of the association between regulator of telomere elongation helicase 1 rs6010620 polymorphism and glioma through a meta-analysis. Methods: The meta-analysis included 19 published case-control studies involving 8541 cases and 14226 controls. The included papers were searched from PubMed and Embase database. Odds ratio (OR) with 95% confidence interval (95% CI) were used to evaluate the association of regulator of telomere elongation helicase 1 rs6010620 polymorphism with glioma. Results: A significant association between regulator of telomere elongation helicase 1 rs6010620 polymorphism and glioma susceptibility was observed for GG vs. AA+AG (OR=1.28, 95% CI=1.14-1.43) and G vs. A (OR=1.07, 95% CI=1.03-1.10). Further subgroup analysis based on ethnicity showed similar results in Asians and Caucasians. In the subgroup analysis of source of control, a significant association between the G allele and glioma susceptibility were found in population-based group and hospital-based group. Conclusions: The meta-analysis suggested that regulator of telomere elongation helicase 1 rs6010620 polymorphism was a risk factor for glioma. And this study also suggested that rs6010620 GG genotype and G allele may be indicators for the risk of glioma. PMID:25785045

  4. Analysis of perspective elongation for sodium laser guide star

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Zhang, Wei; Chen, Tianjiang; Zhou, Wenchao; Yan, Hong

    2015-10-01

    Laser Guide Star Adaptive Optical systems become an established technique at telescope facilities with large apertures. At these aperture diameters, such as 8m class telescope facilities, the finite distance and vertical extend of an artificial excited guide star result in perspective elongation, which produces errors in wave-front reconstruction and could influence the performance of adaptive optical systems seriously. In this paper, we shall briefly introduce and explain the effect of the perspective elongation, and show some results of theoretical simulation and experiment. First of all, we analyzed how the perspective elongation of sodium LGS changes, and gave the results of simulation which indicated the relation between the perspective elongation and some related parameters. The aberration caused by the elongation was analyzed, and the possibility of aberration correction was discussed. Based on the results of the theoretical simulation, we designed an experiment to observe the perspective elongation. A transmitting and receiving system has been set up. The system consisted of a 300mJ sodium LGS laser, a telescope with an aperture diameter of 450mm, a beam expander with an aperture diameter of 200mm, a LGS detecting device, etc. Based on the pulsed laser and the mobile LGS projector, we operated the experiment at different distance between the telescope and the laser projector. A series of elongated images, corresponding the distance from 5m to 30m, was obtained. The analytic results of the image data agreed with the theoretical simulation. Based on the experimental data, we deduced the aberration of wave-front at 30m separation. According to theoretical simulation of the perspective elongation, the effects including the aberration of wave-front could be achieved, which had been partially verified in the experiment. We suggest that one could improve the reconstruction accuracy in a sodium or Rayleigh LGS adaptive optical system by eliminating the influence of

  5. Multiple P-TEFbs cooperatively regulate the release of promoter-proximally paused RNA polymerase II.

    PubMed

    Lu, Xiaodong; Zhu, Xinxing; Li, You; Liu, Min; Yu, Bin; Wang, Yu; Rao, Muhua; Yang, Haiyang; Zhou, Kai; Wang, Yao; Chen, Yanheng; Chen, Meihua; Zhuang, Songkuan; Chen, Lin-Feng; Liu, Runzhong; Chen, Ruichuan

    2016-08-19

    The association of DSIF and NELF with initiated RNA Polymerase II (Pol II) is the general mechanism for inducing promoter-proximal pausing of Pol II. However, it remains largely unclear how the paused Pol II is released in response to stimulation. Here, we show that the release of the paused Pol II is cooperatively regulated by multiple P-TEFbs which are recruited by bromodomain-containing protein Brd4 and super elongation complex (SEC) via different recruitment mechanisms. Upon stimulation, Brd4 recruits P-TEFb to Spt5/DSIF via a recruitment pathway consisting of Med1, Med23 and Tat-SF1, whereas SEC recruits P-TEFb to NELF-A and NELF-E via Paf1c and Med26, respectively. P-TEFb-mediated phosphorylation of Spt5, NELF-A and NELF-E results in the dissociation of NELF from Pol II, thereby transiting transcription from pausing to elongation. Additionally, we demonstrate that P-TEFb-mediated Ser2 phosphorylation of Pol II is dispensable for pause release. Therefore, our studies reveal a co-regulatory mechanism of Brd4 and SEC in modulating the transcriptional pause release by recruiting multiple P-TEFbs via a Mediator- and Paf1c-coordinated recruitment network. PMID:27353326

  6. Pausing on Polyribosomes: Make Way for Elongation in Translational Control.

    PubMed

    Richter, Joel D; Coller, Jeff

    2015-10-01

    Among the three phases of mRNA translation-initiation, elongation, and termination-initiation has traditionally been considered to be rate limiting and thus the focus of regulation. Emerging evidence, however, demonstrates that control of ribosome translocation (polypeptide elongation) can also be regulatory and indeed exerts a profound influence on development, neurologic disease, and cell stress. The correspondence of mRNA codon usage and the relative abundance of their cognate tRNAs is equally important for mediating the rate of polypeptide elongation. Here, we discuss recent results showing that ribosome pausing is a widely used mechanism for controlling translation and, as a result, biological transitions in health and disease.

  7. Histone chaperones Nap1 and Vps75 regulate histone acetylation during transcription elongation.

    PubMed

    Xue, Yu-Ming; Kowalska, Anna K; Grabowska, Kamila; Przybyt, Katarzyna; Cichewicz, Magda A; Del Rosario, Brian C; Pemberton, Lucy F

    2013-04-01

    Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation. PMID:23401858

  8. A Pollen-Specific RALF from Tomato That Regulates Pollen Tube Elongation12[W][OA

    PubMed Central

    Covey, Paul A.; Subbaiah, Chalivendra C.; Parsons, Ronald L.; Pearce, Gregory; Lay, Fung T.; Anderson, Marilyn A.; Ryan, Clarence A.; Bedinger, Patricia A.

    2010-01-01

    Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 μm peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 μm in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window. PMID:20388667

  9. Arabidopsis thaliana root elongation growth is sensitive to lunisolar tidal acceleration and may also be weakly correlated with geomagnetic variations

    PubMed Central

    Barlow, Peter W.; Fisahn, Joachim; Yazdanbakhsh, Nima; Moraes, Thiago A.; Khabarova, Olga V.; Gallep, Cristiano M.

    2013-01-01

    Background Correlative evidence suggests a relationship between the lunisolar tidal acceleration and the elongation rate of arabidopsis roots grown under free-running conditions of constant low light. Methods Seedlings of Arabidopsis thaliana were grown in a controlled-climate chamber maintained at a constant temperature and subjected to continuous low-level illumination from fluorescent tubes, conditions that approximate to a ‘free-running’ state in which most of the abiotic factors that entrain root growth rates are excluded. Elongation of evenly spaced, vertical primary roots was recorded continuously over periods of up to 14 d using high temporal- and spatial-resolution video imaging and were analysed in conjunction with geophysical variables. Key Results and Conclusions The results confirm the lunisolar tidal/root elongation relationship. Also presented are relationships between the hourly elongation rates and the contemporaneous variations in geomagnetic activity, as evaluated from the disturbance storm time and ap indices. On the basis of time series of root elongation rates that extend over ≥4 d and recorded at different seasons of the year, a provisional conclusion is that root elongation responds to variation in the lunisolar force and also appears to adjust in accordance with variations in the geomagnetic field. Thus, both lunisolar tidal acceleration and the geomagnetic field should be considered as modulators of root growth rate, alongside other, stronger and more well-known abiotic environmental regulators, and perhaps unexplored factors such as air ions. Major changes in atmospheric pressure are not considered to be a factor contributing to oscillations of root elongation rate. PMID:23532042

  10. Non-centrosomal epidermal microtubules act in parallel to LET-502/ROCK to promote C. elegans elongation.

    PubMed

    Quintin, Sophie; Wang, Shahoe; Pontabry, Julien; Bender, Ambre; Robin, François; Hyenne, Vincent; Landmann, Frédéric; Gally, Christelle; Oegema, Karen; Labouesse, Michel

    2016-01-01

    C. elegans embryonic elongation is a morphogenetic event driven by actomyosin contractility and muscle-induced tension transmitted through hemidesmosomes. A role for the microtubule cytoskeleton has also been proposed, but its contribution remains poorly characterized. Here, we investigate the organization of the non-centrosomal microtubule arrays present in the epidermis and assess their function in elongation. We show that the microtubule regulators γ-tubulin and NOCA-1 are recruited to hemidesmosomes and adherens junctions early in elongation. Several parallel approaches suggest that microtubule nucleation occurs from these sites. Disrupting the epidermal microtubule array by overexpressing the microtubule-severing protein Spastin or by inhibiting the C. elegans ninein homolog NOCA-1 in the epidermis mildly affected elongation. However, microtubules were essential for elongation when hemidesmosomes or the activity of the Rho kinase LET-502/ROCK were partially compromised. Imaging of junctional components and genetic analyses suggest that epidermal microtubules function together with Rho kinase to promote the transport of E-cadherin to adherens junctions and myotactin to hemidesmosomes. Our results indicate that the role of LET-502 in junctional remodeling is likely to be independent of its established function as a myosin II activator, but requires a microtubule-dependent pathway involving the syntaxin SYX-5. Hence, we propose that non-centrosomal microtubules organized by epidermal junctions contribute to elongation by transporting junction remodeling factors, rather than having a mechanical role.

  11. Analysis of changes in relative elemental growth rate patterns in the elongation zone of Arabidopsis roots upon gravistimulation

    NASA Technical Reports Server (NTRS)

    Mullen, J. L.; Ishikawa, H.; Evans, M. L.

    1998-01-01

    Although Arabidopsis is an important system for studying root physiology, the localized growth patterns of its roots have not been well defined, particularly during tropic responses. In order to characterize growth rate profiles along the apex of primary roots of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) we applied small charcoal particles to the root surface and analyzed their displacement during growth using an automated video digitizer system with custom software for tracking the markers. When growing vertically, the maximum elongation rate occurred 481 +/- 50 microns back from the extreme tip of the root (tip of root cap), and the elongation zone extended back to 912 +/- 137 microns. The distal elongation zone (DEZ) has previously been described as the apical region of the elongation zone in which the relative elemental growth rate (REGR) is < or = 30% of the peak rate in the central elongation zone. By this definition, our data indicate that the basal limit of the DEZ was located 248 +/- 30 microns from the root tip. However, after gravistimulation, the growth patterns of the root changed. Within the first hour of graviresponse, the basal limit of the DEZ and the position of peak REGR shifted apically on the upper flank of the root. This was due to a combination of increased growth in the DEZ and growth inhibition in the central elongation zone. On the lower flank, the basal limit of the DEZ shifted basipetally as the REGR decreased. These factors set up the gradient of growth rate across the root, which drives curvature.

  12. Arabidopsis MICROTUBULE DESTABILIZING PROTEIN40 Is Involved in Brassinosteroid Regulation of Hypocotyl Elongation[C][W][OA

    PubMed Central

    Wang, Xianling; Zhang, Jin; Yuan, Ming; Ehrhardt, David W.; Wang, Zhiyong; Mao, Tonglin

    2012-01-01

    The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana MICROTUBULE DESTABILIZING PROTEIN40 (MDP40) as a positive regulator of hypocotyl cell elongation. BRASSINAZOLE-RESISTANT1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation. PMID:23115248

  13. Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-β and elongated cell shape.

    PubMed

    Wang, Wenbo; Li, Jie; Wang, Keyun; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Ye, Mingliang; Zou, Hanfa; Liu, Wei

    2016-03-01

    Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling. PMID:26632599

  14. Prevalence of Elongated Styloid Process in a Central Brazilian Population

    PubMed Central

    Vieira, Evanice Menezes Marçal; Morais, Sylvania De; Musis, Carlo Ralph De; Albuquerque, Paulo Artur Andrade De; Borges, Álvaro Henrique

    2015-01-01

    Background Eagle’s syndrome comprises a rare disorder caused by compression of an elongated or deformed styloid process or ossified/calcified stylohyoid ligament on neural and vascular structures. It is characterized by facial and neck pain and can be confused with a wide variety of facial neuralgias, oral and dental diseases and temporomandibular disorders. An imaging evaluation associated with a careful clinical examination, are mandatory in structuring a correct differential diagnosis and in the establishment of a proper therapeutic protocol. Aim To investigate the prevalence of the elongated styloid process in a Central Brazilian population and its relation to gender, age and side. Materials and Methods Digital panoramic radiographs of 736 patients (412 female and 324 male, with a mean age of 35.03 years) were consecutively selected from a private radiology clinic’s secondary database. The apparent length of the styloid process was measured from the point where the styloid left the tympanic plate to the tip of the process by two specialists in dental radiology, with the help of the measuring tools on the accompanying software. Styloid process measuring more than 30 mm was considered elongated. The statistical analysis included frequency distribution and cross tabulation. The data were analysed by using Chi-squared tests. The level of significance was set at 5% for all analyses. Results A total of 323 (43.89%) radiographic images were suggestive of elongated styloid process. No statistically significant difference was found between the genders, although a higher prevalence was noticed in female participants. Approximately, 31% of the elongated styloid process was observed in 18-53-year-old participants (p < 0.05). Two hundred and sixty seven styloid processes (36.28%) were elongated on both right and left sides. Conclusion The prevalence of elongated styloid process was high and no statistically significant correlation was found between the presence of

  15. Fatty acid elongation in yeast--biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.

    PubMed

    Dittrich, F; Zajonc, D; Hühne, K; Hoja, U; Ekici, A; Greiner, E; Klein, H; Hofmann, J; Bessoule, J J; Sperling, P; Schweizer, E

    1998-03-15

    Elongation of long-chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrates malonyl-CoA, NADPH and a medium-chain or long-chain acyl-CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12-14 carbon atoms, while shorter-chain-length acyl-CoA were almost (octanoyl-CoA) or completely (hexanoyl-CoA, acetyl-CoA) inactive. In particular, acetyl-CoA was inactive as a primer and as extender unit. The Michaelis constants for octanoyl-CoA (0.33 mM), decanoyl-CoA (0.83 mM) lauroyl-CoA (0.05 mM), myristoyl-CoA (0.4 mM) and palmitoyl-CoA (0.13 mM) were determined and were comparable for fatty acid synthesis and elongation. In contrast, the affinity of malonyl-CoA was 17-fold lower for elongation (Km = 0.13 mM) than for the fatty acid synthase (FAS) system. With increasing chain length of the primer (> or = 12:0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenous fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10-20-fold lower than that of de novo fatty acid synthesis. The same elongation activity is observed in respiratory competent and in mitochondrially defective cells. The products of in vitro fatty acid elongation are fatty acids of 15-17 or 22-26 carbon atoms, depending on whether tridecanoyl-CoA or stearoyl-CoA is used as a primer. In vitro, the elongation products are converted in part, by alpha-oxidation, to their odd-chain-length lower homologues or are hydrolyzed to fatty acids. In contrast, no odd-chain-length elongation products or very-long-chain fatty acids (VLCFA) shorter than 26:0 are observed in vivo. Hence, VLCFA synthesis exhibits a higher processivity in vivo than in the cell

  16. Studies on the mechanism of cell elongation in Blepharisma japonicum I. A physiological mechanism how light stimulation evokes cell elongation.

    PubMed

    Ishida, M; Shigenaka, Y; Taneda, K

    1989-10-27

    In a heterotrichous ciliate, Blepharisma japonicum, longitudinal elongation of the cell body is induced by light stimulation (1000 to 3000 lux). This light-induced response was inhibited under the existence of cyclic mononucleotide phosphodiesterase (PDE) antagonists such as papaverine (10(-4)M), theophylline (10(-3) M), dibutyril-cAMP (10(-4)M), 3-isobutyl-1-methyl-xanthine (10(-4) M) and dibutyril-cGMP (10(-4) M). Microinjection of cyclic mononucleotides, especially cGMP, inhibited cell elongation. These observations suggest that the cell elongation was mediated by intracellular cyclic mononucleotide. K(+) specific ionophore valinomycin (10(-8)-10(-7) M) enhanced light-induced cell elongation. This effect of valinomycin became more remarkable when valinomycin coexisted with PDE antagonists, while it was diminished under high K+ conditions. Moreover, the K(+) channel blockers tetraethylammonium (TEA) and CsCl inhibited cell elongation. These observations suggest that the cell elongation is also mediated by K(+) hyperpolarization, and that this electrical change is probably elicited after the intracellular concentration of cyclic mononucleotide decreased.

  17. TEFM is a potent stimulator of mitochondrial transcription elongation in vitro.

    PubMed

    Posse, Viktor; Shahzad, Saba; Falkenberg, Maria; Hällberg, B Martin; Gustafsson, Claes M

    2015-03-11

    A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.

  18. Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation

    PubMed Central

    Ishimura, Ryuta; Nagy, Gabor; Dotu, Ivan; Chuang, Jeffrey H; Ackerman, Susan L

    2016-01-01

    Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2nmf205-/- mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNAArgUCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2nmf205-/- mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress. DOI: http://dx.doi.org/10.7554/eLife.14295.001 PMID:27085088

  19. Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation.

    PubMed

    Ishimura, Ryuta; Nagy, Gabor; Dotu, Ivan; Chuang, Jeffrey H; Ackerman, Susan L

    2016-01-01

    Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2(nmf205)(-/-) mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNA(Arg)UCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2(nmf205)(-/-) mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress. PMID:27085088

  20. Control of cell proliferation and elongation by miR396.

    PubMed

    Ercoli, María Florencia; Rojas, Arantxa M L; Debernardi, Juan Manuel; Palatnik, Javier F; Rodriguez, Ramiro E

    2016-06-01

    The combinatory effects of cell proliferation and cell elongation determines the rate at which organs growth. In the root meristematic zone cells both divide and expand, while post-mitotic cells in the elongation zone only expands until they reach their final size. The transcription factors of the GROWTH-REGULATING FACTOR (GRF) class promote cell proliferation in various plant organs. Their expression is restricted to cells with a high proliferative capacity, yet strong downregulation of the GRF activity compromise the plant survival. Part of expression pattern of the GRFs is ensured by the post-transcriptional repression mediated by the conserved microRNA miR396. Here we show the quantitative effects in root growth caused by GRF depletion in a series of transgenic lines with different miR396 levels. We show that high miRNA levels affect cell elongation and proliferation in roots. Detailed analysis suggests that cell proliferation is restricted due to a reduction in cell cycle speed that might result from defects in the accumulation of mitotic cyclins. The results provide insights into the participation of the miRNA-GRF regulatory network in root development. PMID:27172373

  1. c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells.

    PubMed

    Gil, Germán A; Bussolino, Daniela F; Portal, Maximiliano M; Alfonso Pecchio, Adolfo; Renner, Marianne L; Borioli, Graciela A; Guido, Mario E; Caputto, Beatriz L

    2004-04-01

    We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation. PMID:14767061

  2. Numerical experiments with flows of elongated granules

    NASA Technical Reports Server (NTRS)

    Elrod, Harold G.; Brewe, David E.

    1992-01-01

    Theory and numerical results are given for a program simulating two dimensional granular flow (1) between two infinite, counter-moving, parallel, roughened walls, and (2) for an infinitely wide slider. Each granule is simulated by a central repulsive force field ratcheted with force restitution factor to introduce dissipation. Transmission of angular momentum between particles occurs via Coulomb friction. The effect of granular hardness is explored. Gaps from 7 to 28 particle diameters are investigated, with solid fractions ranging from 0.2 to 0.9. Among features observed are: slip flow at boundaries, coagulation at high densities, and gross fluctuation in surface stress. A videotape has been prepared to demonstrate the foregoing effects.

  3. RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress.

    PubMed

    Saponaro, Marco; Kantidakis, Theodoros; Mitter, Richard; Kelly, Gavin P; Heron, Mark; Williams, Hannah; Söding, Johannes; Stewart, Aengus; Svejstrup, Jesper Q

    2014-05-22

    RECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes. PMID:24836610

  4. Dynamic transcriptional events in embryonic stem cells mediated by the super elongation complex (SEC)

    PubMed Central

    Lin, Chengqi; Garrett, Alexander S.; De Kumar, Bony; Smith, Edwin R.; Gogol, Madelaine; Seidel, Christopher; Krumlauf, Robb; Shilatifard, Ali

    2011-01-01

    Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We identified an essential role for the ELL (eleven–nineteen lysine-rich leukemia gene)/P-TEFb (positive transcription elongation factor)-containing super elongation complex (SEC) in the regulation of gene expression, including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells also identified a requirement for SEC at genes without paused Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals. PMID:21764852

  5. CEP120 interacts with CPAP and positively regulates centriole elongation.

    PubMed

    Lin, Yi-Nan; Wu, Chien-Ting; Lin, Yu-Chih; Hsu, Wen-Bin; Tang, Chieh-Ju C; Chang, Ching-Wen; Tang, Tang K

    2013-07-22

    Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1-associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle-regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding-defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.

  6. An Integrated Approach Reveals Regulatory Controls on Bacterial Translation Elongation

    PubMed Central

    Subramaniam, Arvind R.; Zid, Brian M.; O’Shea, Erin K.

    2014-01-01

    Summary Ribosomes elongate at a non-uniform rate during translation. Theoretical models and experiments disagree on the in vivo determinants of elongation rate and the mechanism by which elongation rate affects protein levels. To resolve this conflict, we measured transcriptome-wide ribosome occupancy under multiple conditions and used it to formulate a whole-cell model of translation in E. coli. Our model predicts that elongation rates at most codons during nutrient-rich growth are not limited by the intracellular concentrations of aminoacyl-tRNAs. However, elongation pausing during starvation for single amino acids is highly sensitive to the kinetics of tRNA aminoacylation. We further show that translation abortion upon pausing accounts for the observed ribosome occupancy along mRNAs during starvation. Abortion reduces global protein synthesis, but it enhances the translation of a subset of mRNAs. These results suggest a regulatory role for aminoacylation and abortion during stress, and our study provides an experimentally-constrained framework for modeling translation. PMID:25416955

  7. Three Cases of Elongated Mandibular Coronoid Process with Different Presentations

    PubMed Central

    Ilguy, Mehmet; Kursoglu, Pinar; Ilguy, Dilhan

    2014-01-01

    Abnormal elongation of the mandibular coronoid process is rare and its etiology is not yet elucidated. The aim of this report is to demonstrate and discuss the relationship between elongated mandibular coronoid process and limitation of mouth opening with cone beam computed tomography. Although the clinical characteristic of elongation of the coronoid process is mandibular limitation, in this report, one case had problem with mouth opening. Axial scans revealed that the distance between the coronoid process and the inner face of the frontal part of the zygomatic bone may cause limitation in mouth opening. In conclusion, instead of the length, the distance between the coronoid process and the inner face of the frontal part of the zygomatic bone may be the actual reason for limitation of mouth opening. This may prevent misdiagnosis. PMID:24693298

  8. Wnt5a is essential for intestinal elongation in mice

    PubMed Central

    Cervantes, Sara; Yamaguchi, Terry P.; Hebrok, Matthias

    2009-01-01

    Summary Morphogenesis of the mammalian small intestine entails extensive elongation and folding of the primitive gut into a tightly coiled digestive tube. Surprisingly, little is known about the cellular and molecular mechanisms that mediate the morphological aspects of small intestine formation. Here, we demonstrate that Wnt5a, a member of the Wnt family of secreted proteins, is essential for the development and elongation of the small intestine from the midgut region. We found that the small intestine in mice lacking Wnt5a was dramatically shortened and duplicated, forming a bifurcated lumen instead of a single tube. In addition, cell proliferation was reduced and re-intercalation of post-mitotic cells into the elongating gut tube epithelium was disrupted. Thus, our study demonstrates that Wnt5a functions as a critical regulator of midgut formation and morphogenesis in mammals. PMID:19100728

  9. An Elongated Tetrakaidecahedron Model for Open-Celled Foams

    NASA Technical Reports Server (NTRS)

    Sullivan, Roy M.; Ghosn, Louis J.; Lerch, Bradley A.

    2007-01-01

    A micro-mechanics model for non-isotropic, open-celled foams is developed using an elongated tetrakaidecahedron (Kelvin model) as the repeating unit cell. The micro-mechanics model employs an elongated Kelvin model geometry which is more general than that employed by previous authors. Assuming the cell edges possess axial and bending rigidity, the mechanics of deformation of the elongated tetrakaidecahedron lead to a set of equations for the Young's modulus, Poisson's ratio and strength of the foam in the principal material directions. These equations are written as a function of the cell edge lengths and cross-section properties, the inclination angle and the strength and stiffness of the solid material. The model is applied to predict the strength and stiffness of several polymeric foams. Good agreement is observed between the model results and the experimental measurements.

  10. Elongate summit calderas as Neogene paleostress indicators in Antarctica

    USGS Publications Warehouse

    Paulsen, T.S.; Wilson, T.J.

    2007-01-01

    The orientations and ages of elongate summit calderas on major polygenetic volcanoes were compiled to document Miocene to Pleistocene Sh (minimum horizontal stress) directions on the western and northern flanks of the West Antarctic rift system. Miocene to Pleistocene summit calderas along the western Ross Sea show relatively consistent ENE long axis trends, which are at a high angle to the Transantarctic Mountain Front and parallel to the N77ºE Sh direction at Cape Roberts. The elongation directions of many Miocene to Pleistocene summit calderas in Marie Byrd Land parallel the alignment of polygenetic volcanoes in which they occur, except several Pleistocene calderas with consistent NNE to NE trends. The overall pattern of elongate calderas in Marie Byrd Land is probably due to a combination of structurally controlled orientations and regional stress fields in which Sh is oriented NNE to NE at a moderate to high angle to the trace of the West Antarctic rift system.

  11. Break-induced replication and recombinational telomere elongation in yeast.

    PubMed

    McEachern, Michael J; Haber, James E

    2006-01-01

    When a telomere becomes unprotected or if only one end of a chromosomal double-strand break succeeds in recombining with a template sequence, DNA can be repaired by a recombination-dependent DNA replication process termed break-induced replication (BIR). In budding yeasts, there are two BIR pathways, one dependent on the Rad51 recombinase protein and one Rad51 independent; these two repair processes lead to different types of survivors in cells lacking the telomerase enzyme that is required for normal telomere maintenance. Recombination at telomeres is triggered by either excessive telomere shortening or disruptions in the function of telomere-binding proteins. Telomere elongation by BIR appears to often occur through a "roll and spread" mechanism. In this process, a telomeric circle produced by recombination at a dysfunctional telomere acts as a template for a rolling circle BIR event to form an elongated telomere. Additional BIR events can then copy the elongated sequence to all other telomeres.

  12. Non-targeted metabolomic evaluation of the uterine milieu during the transitional period of embryo elongation in the pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alterations in the signaling of critical molecular factors within the uterine milieu lead to deficiencies in embryo elongation. The objective of this study was to identify metabolites within the uterine environment that are present as porcine embryos transition between spherical, ovoid, and tubular ...

  13. River Elongation as a Proxy for Lateral Channel Activity

    NASA Astrophysics Data System (ADS)

    Lauer, J. W.

    2009-12-01

    Lateral channel movement is a process that is tightly linked to both channel hydraulics and sediment transport, strongly influences floodplain ecology, and also has great relevance for banktop property owners. The correlation between channel migration rate and channel curvature usually causes meandering river channels to elongate as they migrate laterally. Over the long term, the increase in sinuosity is compensated by a rapid decrease in sinuosity where and when river bends shorten through cutoff processes. However, the elongation for most meander bends in systems free to migrate across wide floodplains often occurs relatively uniformly throughout the system. Consequently, the rate of elongation of individual river bends, integrated across a river reach, offers a simple mechanism for characterizing the reach’s lateral activity. Spatial series of accumulated elongation can also be used to delineate reaches with similar properties. We use aerial imagery pairs to compare rates of lateral channel centerline shifting with channel centerline elongation for reaches many bends long along eight different rivers with widths ranging from 12 to 584 m. Except where bends translate downstream without changing form, elongation rates are closely linked to lateral shifting. In several cases, a change in elongation rate corresponds closely with a change in channel width, discharge, and/or bed material. For reaches free to migrate across a wide, unconfined floodplain and where lateral migration measurements are likely of high quality, the average ratio between the reach average migration rate normalized by channel width and the rate of sinuosity increase (excluding bends that experienced a cutoff between imagery dates) is approximately 5.6. Since elongation rate measurements can be made accurately even from photos that are poorly aligned, the relationship between sinuosity increase and lateral migration potentially provides a means of bypassing time-consuming georeferencing

  14. Rotation in Free Fall of Rectangular Wings of Elongated Shape

    NASA Technical Reports Server (NTRS)

    Dupleich, Paul

    1949-01-01

    The present report of Mr. Dupleich is the summary of a very extensive experimental study of the well-known mechanical phenomenon: the rotation in free fall (* air, for instance) of more or less elongated rectangles cut out of paper or pasteboard. This phenomenon, the conditions for existence of which depend chiefly on the elongated of the small plate and its weight per unit area, is essentially an aerodynamic phenomenon and as such, raises questions of a certain interest to our department.We believe that the modern concepts of the mechanics of fluids do not have the range attributed to them.

  15. Correlations between changes in electrical parameters and changes in cell elongation rates in gravistimulated roots.

    PubMed

    Ishikawa, H; Evans, M L

    1994-01-01

    The earliest changes in growth rate following the gravistimulation of roots occur in a special group of cells between the meristem and the elongating region of the root. This zone is called the postmitotic isodiametric growth (PIG) zone and consists of cells which have ceased dividing and are expanding isodiametrically. Upon gravistimulation cells along the upper side of the PIG zone begin elongating rapidly and this accounts for much of the early growth asymmetry. There is rapid (< 30 s) hyperpolarization of cells on the upper side of the PIG zone as well as rapid uptake of potassium from the stele. We propose that there is a relationship between the rate of hydrogen ion efflux and the extent of membrane hyperpolarization in the PIG zone and that such changes in potential are an early indication of impending changes in growth performance. Although the development of auxin asymmetry in the cap and its transmission to the elongating region is considered to be the controlling factor in root gravitropism, auxin asymmetry in the cap develops only after 30 min, about the same as the lag before initiation of curvature. Although this dilemma may be partly resolved by the location of the PIG zone close to the cap, alternative explanations such as gravi-detection by the PIG zone or very rapid (electrical?) signal transmission from the cap to the PIG zone need to be considered. PMID:11537910

  16. Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana.

    PubMed

    Oh, Sookyung; Warnasooriya, Sankalpi N; Montgomery, Beronda L

    2013-04-01

    Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.

  17. NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis

    PubMed Central

    Dolata, Jakub; Guo, Yanwu; Kołowerzo, Agnieszka; Smoliński, Dariusz; Brzyżek, Grzegorz; Jarmołowski, Artur; Świeżewski, Szymon

    2015-01-01

    The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5′ and 3′ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis. PMID:25568310

  18. VAMP-2 promotes neurite elongation and SNAP-25A increases neurite sprouting in PC12 cells.

    PubMed

    Shirasu, M; Kimura, K; Kataoka, M; Takahashi, M; Okajima, S; Kawaguchi, S; Hirasawa, Y; Ide, C; Mizoguchi, A

    2000-08-01

    Recent studies suggest that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion in the plasma membrane that allows expansion for neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. In this study, we took the opposite approach by overexpressing green fluorescent protein (GFP)-fusion SNAREs, including VAMP-2, SNAP-25A, and syntaxin1A, in PC12 cells to investigate the role of SNAREs in the neurite outgrowth of PC12 cells. Neurite outgrowth analysis demonstrated that: (1) GFP-VAMP-2 increased the length of individual neurites, without changing the number of neurites per cell; (2) GFP-SNAP-25A increased the number of neurites per cell, with no change in the length of the individual neurites. In both cases, the total length of neurites per cell was increased; (3) GFP-syntaxin1A resulted in no significant change, either in neurite length, or in the number of neurites per cell. These findings suggest that when overexpressed in PC12 cells, VAMP-2 can promote neurite elongation, while SNAP-25A can stimulate neurite sprouting. On the other hand, overexpression of syntaxin1A neither promotes nor inhibits neurite outgrowth. Thus VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.

  19. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation

    SciTech Connect

    Thompson, Morgan E; Heimsath, Ernest G; Gauvin, Timothy J; Higgs, Henry N; Kull, F Jon

    2012-12-09

    Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

  20. Molecular development of the mid-stage elongating cotton fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton fiber is one of the leading natural textile fibers and is the leading value added crop in the USA. The annual business revenue from the cotton industry exceeds $120 billion. The growth of the cotton fiber is divided into four unique, yet overlapping stages; initiation, elongation, secondary w...

  1. FtsZ-Dependent Elongation of a Coccoid Bacterium

    PubMed Central

    Pereira, Ana R.; Hsin, Jen; Król, Ewa; Tavares, Andreia C.; Flores, Pierre; Hoiczyk, Egbert; Ng, Natalie; Dajkovic, Alex; Brun, Yves V.; VanNieuwenhze, Michael S.; Roemer, Terry; Carballido-Lopez, Rut; Huang, Kerwyn Casey

    2016-01-01

    ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZG193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZG193D filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. PMID:27601570

  2. Fiber elongation-dependent aquaporin expression in different cotton cultivars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fiber length is an important component to measure cotton yield potential. Many research efforts have focused on development of cotton cultivars with higher fiber quality and yield potential. It is known that genetic diversity exists for fiber elongation. Understanding the genetic and molecular mecha...

  3. Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

    PubMed Central

    Wu, Zhe; Ietswaart, Robert; Liu, Fuquan; Yang, Hongchun; Howard, Martin; Dean, Caroline

    2016-01-01

    The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼25-fold with elongation rate varying ∼8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context. PMID:26699513

  4. STRIP BEING REDUCED IN CROSSSECTION AND ELONGATED ON RUNOUT LINE ...

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

    STRIP BEING REDUCED IN CROSS-SECTION AND ELONGATED ON RUN-OUT LINE OF #43 HOT MILL. MECHANICAL TRANSFER HAS MOVED INTO POSITION OVER FURNACE DISCHARGE LINE (L) TO RETRIEVE ANOTHER CAKE. - American Brass Foundry, 70 Sayre Street, Buffalo, Erie County, NY

  5. Quadratic elongation: A quantitative measure of distortion in coordination polyhedra

    USGS Publications Warehouse

    Robinson, Kelly F.; Gibbs, G.V.; Ribbe, P.H.

    1971-01-01

    Quadratic elongation and the variance of bond angles are linearly correlated for distorted octahedral and tetrahedral coordination complexes, both of which show variations in bond length and bond angle. The quadratic elonga tion is dimensionless, giving a quantitative measure of polyhedral distortion which is independent of the effective size of the polyhedron.

  6. Collisional diffusion in toroidal plasmas with elongation and triangularity

    SciTech Connect

    Martin, P.; Castro, E.; Haines, M. G.

    2007-05-15

    Collisional diffusion is analyzed for plasma tokamaks with different ellipticities and triangularities. Improved nonlinear equations for the families of magnetic surfaces are used here. Dimensionless average velocities are calculated as a function of the inductive electric field, elongation, triangularity, and Shafranov shift. Confinement has been found to depend significantly on triangularity.

  7. Binary asteroid population. 3. Secondary rotations and elongations

    NASA Astrophysics Data System (ADS)

    Pravec, P.; Scheirich, P.; Kušnirák, P.; Hornoch, K.; Galád, A.; Naidu, S. P.; Pray, D. P.; Világi, J.; Gajdoš, Š.; Kornoš, L.; Krugly, Yu. N.; Cooney, W. R.; Gross, J.; Terrell, D.; Gaftonyuk, N.; Pollock, J.; Husárik, M.; Chiorny, V.; Stephens, R. D.; Durkee, R.; Reddy, V.; Dyvig, R.; Vraštil, J.; Žižka, J.; Mottola, S.; Hellmich, S.; Oey, J.; Benishek, V.; Kryszczyńska, A.; Higgins, D.; Ries, J.; Marchis, F.; Baek, M.; Macomber, B.; Inasaridze, R.; Kvaratskhelia, O.; Ayvazian, V.; Rumyantsev, V.; Masi, G.; Colas, F.; Lecacheux, J.; Montaigut, R.; Leroy, A.; Brown, P.; Krzeminski, Z.; Molotov, I.; Reichart, D.; Haislip, J.; LaCluyze, A.

    2016-03-01

    We collected data on rotations and elongations of 46 secondaries of binary and triple systems among near-Earth, Mars-crossing and small main belt asteroids. 24 were found or are strongly suspected to be synchronous (in 1:1 spin-orbit resonance), and the other 22, generally on more distant and/or eccentric orbits, were found or are suggested to have asynchronous rotations. For 18 of the synchronous secondaries, we constrained their librational angles, finding that their long axes pointed to within 20° of the primary on most epochs. The observed anti-correlation of secondary synchroneity with orbital eccentricity and the limited librational angles agree with the theories by Ćuk and Nesvorný (Ćuk, M., Nesvorný, D. [2010]. Icarus 207, 732-743) and Naidu and Margot (Naidu, S.P., Margot, J.-L. [2015]. Astron. J. 149, 80). A reason for the asynchronous secondaries being on wider orbits than synchronous ones may be longer tidal circularization time scales at larger semi-major axes. The asynchronous secondaries show relatively fast spins; their rotation periods are typically < 10 h. An intriguing observation is a paucity of chaotic secondary rotations; with an exception of (35107) 1991 VH, the secondary rotations are single-periodic with no signs of chaotic rotation and their periods are constant on timescales from weeks to years. The secondary equatorial elongations show an upper limit of a2 /b2 ∼ 1.5 . The lack of synchronous secondaries with greater elongations appears consistent, considering uncertainties of the axis ratio estimates, with the theory by Ćuk and Nesvorný that predicts large regions of chaotic rotation in the phase space for a2 /b2 ≳√{ 2 } . Alternatively, secondaries may not form or stay very elongated in gravitational (tidal) field of the primary. It could be due to the secondary fission mechanism suggested by Jacobson and Scheeres (Jacobson, S.A., Scheeres, D.J. [2011]. Icarus 214, 161-178), as its efficiency is correlated with the

  8. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  9. Stimulation of root elongation and curvature by calcium.

    PubMed

    Takahashi, H; Scott, T K; Suge, H

    1992-01-01

    Ca2+ has been proposed to mediate inhibition of root elongation. However, exogenous Ca2+ at 10 or 20 millimolar, applied directly to the root cap, significantly stimulated root elongation in pea (Pisum sativum L.) and corn (Zea mays L.) seedlings. Furthermore, Ca2+ at 1 to 20 millimolar, applied unilaterally to the caps of Alaska pea roots, caused root curvature away from the Ca2+ source, which was caused by an acceleration of elongation growth on the convex side (Ca2+ side) of the roots. Roots of an agravitropic pea mutant, ageotropum, responded to a greater extent. Roots of Merit and Silver Queen corn also responded to Ca2+ in similar ways but required a higher Ca2+ concentration than that of pea roots. Roots of all other cultivars tested (additional four cultivars of pea and one of corn) curved away from the unilateral Ca2+ source as well. The Ca(2+)-stimulated curvature was substantially enhanced by light. A Ca2+ ionophore, A23187, at 20 micromolar or abscisic acid at 0.1 to 100 micromolar partially substituted for the light effect and enhanced the Ca(2+)-stimulated curvature in the dark. Unilateral application of Ca2+ to the elongation zone of intact roots or to the cut end of detipped roots caused either no curvature or very slight curvature toward the Ca2+. Thus, Ca2+ action on root elongation differs depending on its site of application. The stimulatory action of Ca2+ may involve an elevation of cytoplasmic Ca2+ in root cap cells and may partipate in root tropisms. PMID:11537880

  10. Stimulation of root elongation and curvature by calcium

    NASA Technical Reports Server (NTRS)

    Takahashi, H.; Scott, T. K.; Suge, H.

    1992-01-01

    Ca2+ has been proposed to mediate inhibition of root elongation. However, exogenous Ca2+ at 10 or 20 millimolar, applied directly to the root cap, significantly stimulated root elongation in pea (Pisum sativum L.) and corn (Zea mays L.) seedlings. Furthermore, Ca2+ at 1 to 20 millimolar, applied unilaterally to the caps of Alaska pea roots, caused root curvature away from the Ca2+ source, which was caused by an acceleration of elongation growth on the convex side (Ca2+ side) of the roots. Roots of an agravitropic pea mutant, ageotropum, responded to a greater extent. Roots of Merit and Silver Queen corn also responded to Ca2+ in similar ways but required a higher Ca2+ concentration than that of pea roots. Roots of all other cultivars tested (additional four cultivars of pea and one of corn) curved away from the unilateral Ca2+ source as well. The Ca(2+)-stimulated curvature was substantially enhanced by light. A Ca2+ ionophore, A23187, at 20 micromolar or abscisic acid at 0.1 to 100 micromolar partially substituted for the light effect and enhanced the Ca(2+)-stimulated curvature in the dark. Unilateral application of Ca2+ to the elongation zone of intact roots or to the cut end of detipped roots caused either no curvature or very slight curvature toward the Ca2+. Thus, Ca2+ action on root elongation differs depending on its site of application. The stimulatory action of Ca2+ may involve an elevation of cytoplasmic Ca2+ in root cap cells and may partipate in root tropisms.

  11. Research on the processing technology of elongated holes based on rotary ultrasonic drilling

    NASA Astrophysics Data System (ADS)

    Tong, Yi; Chen, Jianhua; Sun, Lipeng; Yu, Xin; Wang, Xin

    2014-08-01

    The optical glass is hard, brittle and difficult to process. Based on the method of rotating ultrasonic drilling, the study of single factor on drilling elongated holes was made in optical glass. The processing equipment was DAMA ultrasonic machine, and the machining tools were electroplated with diamond. Through the detection and analysis on the processing quality and surface roughness, the process parameters (the spindle speed, amplitude, feed rate) of rotary ultrasonic drilling were researched, and the influence of processing parameters on surface roughness was obtained, which will provide reference and basis for the actual processing.

  12. Quantifying elongation rhythm during full-length protein synthesis.

    PubMed

    Rosenblum, Gabriel; Chen, Chunlai; Kaur, Jaskiran; Cui, Xiaonan; Zhang, Haibo; Asahara, Haruichi; Chong, Shaorong; Smilansky, Zeev; Goldman, Yale E; Cooperman, Barry S

    2013-07-31

    Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification. PMID:23822614

  13. Elongated solid electrolyte cell configurations and flexible connections therefor

    DOEpatents

    Reichner, Philip

    1989-01-01

    A flexible, high temperature, solid oxide electrolyte electrochemical cell stack configuration is made, comprising a plurality of flattened, elongated, connected cell combinations 1, each cell combination containing an interior electrode 2 having a top surface and a plurality of interior gas feed conduits 3, through its axial length, electrolyte 5 contacting the interior electrode and exterior electrode 8 contacting electrolyte, where a major portion of the air electrode top surface 7 is covered by interconnection material 6, and where each cell has at least one axially elongated, electronically conductive, flexible, porous, metal fiber felt material 9 in electronic connection with the air electrode 2 through contact with a major portion of the interconnection material 6, the metal fiber felt being effective as a shock absorbent body between the cells.

  14. Mapping the Escherichia coli transcription elongation complex with exonuclease III

    PubMed Central

    Liu, Zhaokun; Artsimovitch, Irina

    2014-01-01

    Summary RNA polymerase interactions with the nucleic acids control every step of the transcription cycle. These contacts mediate RNA polymerase recruitment to promoters; induce pausing during RNA chain synthesis, and control transcription termination. These interactions are dissected using footprinting assays, in which a bound protein protects nucleic acids from the digestion by nucleases or modification by chemical probes. Exonuclease III is frequently employed to study protein-DNA interactions owing to relatively simple procedures and low background. Exonuclease III has been used to determine RNA polymerase position in transcription initiation and elongation complexes and to infer the translocation register of the enzyme. In this chapter, we describe probing the location and the conformation of transcription elongation complexes formed by walking of the RNA polymerase along an immobilized template. PMID:25665555

  15. DNA sequencing by synthesis based on elongation delay detection

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2015-03-01

    The one of most important problem in modern genetics, biology and medicine is determination of the primary nucleotide sequence of the DNA of living organisms (DNA sequencing). This paper describes the label-free DNA sequencing approach, based on the observation of a discrete dynamics of DNA sequence elongation phase. The proposed DNA sequencing principle are studied by numerical simulation. The numerical model for proposed label-free DNA sequencing approach is based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) and dynamics of nucleotides incorporation to rising DNA strand. The estimates for number of copied DNA sequences for required probability of nucleotide incorporation event detection and correct DNA sequence determination was obtained. The proposed approach can be applied at all known DNA sequencing devices with "sequencing by synthesis" principle of operation.

  16. Elongated solid electrolyte cell configurations and flexible connections therefor

    DOEpatents

    Reichner, P.

    1989-10-17

    A flexible, high temperature, solid oxide electrolyte electrochemical cell stack configuration is made, comprising a plurality of flattened, elongated, connected cell combinations, each cell combination containing an interior electrode having a top surface and a plurality of interior gas feed conduits, through its axial length, electrolyte contacting the interior electrode and exterior electrode contacting electrolyte, where a major portion of the air electrode top surface is covered by interconnection material, and where each cell has at least one axially elongated, electronically conductive, flexible, porous, metal fiber felt material in electronic connection with the air electrode through contact with a major portion of the interconnection material, the metal fiber felt being effective as a shock absorbent body between the cells. 4 figs.

  17. TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

    PubMed

    Moravec, Martin; Wischnewski, Harry; Bah, Amadou; Hu, Yan; Liu, Na; Lafranchi, Lorenzo; King, Megan C; Azzalin, Claus M

    2016-07-01

    Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres.

  18. New Insights on Plant Cell Elongation: A Role for Acetylcholine

    PubMed Central

    Di Sansebastiano, Gian-Pietro; Fornaciari, Silvia; Barozzi, Fabrizio; Piro, Gabriella; Arru, Laura

    2014-01-01

    We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response. PMID:24642879

  19. New insights on plant cell elongation: a role for acetylcholine.

    PubMed

    Di Sansebastiano, Gian-Pietro; Fornaciari, Silvia; Barozzi, Fabrizio; Piro, Gabriella; Arru, Laura

    2014-01-01

    We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response. PMID:24642879

  20. Neuroprotective Copper Bis(thiosemicarbazonato) Complexes Promote Neurite Elongation

    PubMed Central

    Bica, Laura; Liddell, Jeffrey R.; Donnelly, Paul S.; Duncan, Clare; Caragounis, Aphrodite; Volitakis, Irene; Paterson, Brett M.; Cappai, Roberto; Grubman, Alexandra; Camakaris, James; Crouch, Peter J.; White, Anthony R.

    2014-01-01

    Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, CuII(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that CuII(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, CuII(atsm), but at a higher concentration. Induction of neurite elongation by CuII(gtsm) was restricted to neurites within the length range of 75–99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM CuII(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that CuII(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM CuII(gtsm), suggesting a potential link between CuII(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes. PMID:24587210

  1. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  2. Cloning and Expression Analysis of Vvlcc3, a Novel and Functional Laccase Gene Possibly Involved in Stipe Elongation

    PubMed Central

    Lu, Yuanping; Wu, Guangmei; Lian, Lingdan; Guo, Lixian; Wang, Wei; Yang, Zhiyun; Miao, Juan; Chen, Bingzhi; Xie, Baogui

    2015-01-01

    Volvariella volvacea, usually harvested in its egg stage, is one of the most popular mushrooms in Asia. The rapid transition from the egg stage to elongation stage, during which the stipe stretches to almost full length leads to the opening of the cap and rupture of the universal veil, and is considered to be one of the main factors that negatively impacts the yield and value of V. volvacea. Stipe elongation is a common phenomenon in mushrooms; however, the mechanisms, genes and regulation involved in stipe elongation are still poorly understood. In order to study the genes related to the stipe elongation, we analyzed the transcription of laccase genes in stipe tissue of V. volvacea, as some laccases have been suggested to be involved in stipe elongation in Flammulina velutipes. Based on transcription patterns, the expression of Vvlcc3 was found to be the highest among the 11 laccase genes. Moreover, phylogenetic analysis showed that VvLCC3 has a high degree of identity with other basidiomycete laccases. Therefore, we selected and cloned a laccase gene, named Vvlcc3, a cDNA from V. volvacea, and expressed the cDNA in Pichia pastoris. The presence of the laccase signature L1-L4 on the deduced protein sequence indicates that the gene encodes a laccase. Phylogenetic analysis showed that VvLCC3 clusters with Coprinopsis cinerea laccases. The ability to catalyze ABTS (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation proved that the product of the Vvlcc3 gene was a functional laccase. We also found that the expression of the Vvlcc3 gene in V. volvacea increased during button stage to the elongation stage; it reached its peak in the elongation stage, and then decreased in the maturation stage, which was similar to the trend in the expression of Fv-lac3 and Fv-lac5 in F. velutipes stipe tissue. The similar trend in expression level of these laccase genes of F. velutipes suggested that this gene could be involved in stipe elongation in V. volvacea. PMID

  3. Mechanism of gibberellin-dependent stem elongation in peas

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.; Sovonick-Dunford, S. A.

    1989-01-01

    Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process.

  4. Single-Plane Magnetically Focused Elongated Small Field Proton Beams.

    PubMed

    McAuley, Grant A; Slater, James M; Wroe, Andrew J

    2015-08-01

    We previously performed Monte Carlo simulations of magnetically focused proton beams shaped by a single quadrapole magnet and thereby created narrow elongated beams with superior dose delivery characteristics (compared to collimated beams) suitable for targets of similar geometry. The present study seeks to experimentally validate these simulations using a focusing magnet consisting of 24 segments of samarium cobalt permanent magnetic material adhered into a hollow cylinder. Proton beams with properties relevant to clinical radiosurgery applications were delivered through the magnet to a water tank containing a diode detector or radiochromic film. Dose profiles were analyzed and compared with analogous Monte Carlo simulations. The focused beams produced elongated beam spots with high elliptical symmetry, indicative of magnet quality. Experimental data showed good agreement with simulations, affirming the utility of Monte Carlo simulations as a tool to model the inherent complexity of a magnetic focusing system. Compared to target-matched unfocused simulations, focused beams showed larger peak to entrance ratios (26% to 38%) and focused simulations showed a two-fold increase in beam delivery efficiency. These advantages can be attributed to the magnetic acceleration of protons in the transverse plane that tends to counteract the particle outscatter that leads to degradation of peak to entrance performance in small field proton beams. Our results have important clinical implications and suggest rare earth focusing magnet assemblies are feasible and could reduce skin dose and beam number while delivering enhanced dose to narrow elongated targets (eg, in and around the spinal cord) in less time compared to collimated beams.

  5. Mechanism of gibberellin-dependent stem elongation in peas.

    PubMed

    Cosgrove, D J; Sovonick-Dunford, S A

    1989-01-01

    Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process. PMID:11537446

  6. Elongation fracture of metals containing pre-introduced secondary defects

    NASA Astrophysics Data System (ADS)

    Arakawa, K.; Ono, K.; Iseki, M.; Kiritani, M.

    2002-01-01

    High-speed, high-strain tensile deformation of thin foils of fcc metals has recently been found to result in the formation of an anomalously high density of small vacancy clusters, probably in the absence of dislocations. In the present study, deformation of secondary-defect-pre-introduced pure Al is performed, at strain rates ranging from 10(-4) to 10(5)/s and a deformation temperature of - 196 or 25 degreesC. Microstructures in the deformed regions are examined by transmission electron microscopy. Vacancy-type dislocation loops and voids are eliminated by the deformation. He-bubbles are observed to elongate under a tensile stress, mainly in the absence of dislocations. Rows of bubbles oriented close to directions of elongation are found, and might result from extreme elongation and division of bubbles. The bubble rows are parallel to particular crystallographic directions, either (001) or (112). This indicates that displacement of atoms during high-speed, high-strain tensile deformation progresses while conforming with the nature of a crystal, even in the absence of dislocations.

  7. A Triple Helix-Loop-Helix/Basic Helix-Loop-Helix Cascade Controls Cell Elongation Downstream of Multiple Hormonal and Environmental Signaling Pathways in Arabidopsis[C][W

    PubMed Central

    Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

    2012-01-01

    Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall–loosening enzymes; HBI1’s DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals. PMID:23221598

  8. T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex.

    PubMed

    Hertweck, Arnulf; Evans, Catherine M; Eskandarpour, Malihe; Lau, Jonathan C H; Oleinika, Kristine; Jackson, Ian; Kelly, Audrey; Ambrose, John; Adamson, Peter; Cousins, David J; Lavender, Paul; Calder, Virginia L; Lord, Graham M; Jenner, Richard G

    2016-06-21

    The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates. PMID:27292648

  9. Initiation and elongation of polyribonucleotide chains on rat ventral-prostate chromatin transcribed by homologous ribonucleic acid polymerase B.

    PubMed Central

    Thomas, P; Davies, P; Griffiths, K

    1977-01-01

    The characteristics of initiation of RNA synthesis and the elongation of RNA chains on rat ventral-prostate chromatin by RNA polymerase B were investigated by two methods. 1. Initiation was carried out under low-salt conditions with three ribonucleoside triphosphates, and elongation was begun in the absence of reinitiation by the addition of the fourth ribonucleoside triphosphate and increasing the salt concentration. 2. Stable initiation complexes were formed by preincubation of enzyme with template at 37 degrees C, elongation was started by the addition of all four ribonucleoside triphosphates and reinitiation or spurious RNA synthesis was prevented by rifamycin AF/013. The latter method gave more reliable results. The dependence of those parameters on the androgenic status of the animal was studied. During the first 24h after castration, elongation was mainly affected, whereas after 72h a smaller number of initiation sites for RNA polymerase B on chromatin was evident. Considerable diurnal variations in the various parameters were observed. Changes in the relative concentrations of the chromatin-associated proteins were also observed after castration. In the rat ventral-prostate gland androgenic steroids may not only influence one stage of the transcriptional process, but may affect many factors involved in the control of gene expression. PMID:562164

  10. Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation1[OPEN

    PubMed Central

    Sun, Jingbo; Ma, Qianqian; Mao, Tonglin

    2015-01-01

    The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation. PMID:26134166

  11. Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation.

    PubMed

    Sun, Jingbo; Ma, Qianqian; Mao, Tonglin

    2015-09-01

    The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation.

  12. Relative Mesothelioma Potencies for Unregulated Respirable Elongated Mineral and Synthetic Particles

    EPA Science Inventory

    For decades uncertainties and contradictions have surrounded the issue of whether exposures to respirable elongated mineral and synthetic particles (REMPs and RESPs) present health risks such as those recognized for exposures to elongated asbestiform mineral particles from the fi...

  13. Studies on the mechanism of cell elongation in Blepharisma japonicum: 3. Cytoplasmic calcium ions may correlate to cell elongation in calmodulin-dependent manner.

    PubMed

    Ishida, M; Shigenaka, Y; Suzaki, T; Taneda, K

    1991-02-22

    Among many heterotrichous ciliates, Blepharisma japonicum especially demonstrates negative phototaxis in response to light stimulation, which is attributed to be caused by swimming acceleration accompanied by cell elongation and ciliary reversal. When Blepharisma cells were treated with 0.1 mM EGTA, cell elongation gradually decreased in its degree as the adaptation time lapsed. Calmodulin inhibitors such as W-7 (10(-5) M) and chlorpromazine (10(-5) M) inhibited cell elongation. These results suggest that a rise in cytoplasmic free Ca(2+) concentration might cause cell elongation through the Ca-calmodulin system. On the other hand, Ca(2+)-channel blockers (La(3+), Co(2+), Cd(2+), Zn(2+), Mn(2+) and verapamil) did not inhibit cell elongation. Ca2+ localization examined by calcium pyroantimonate cytochemistry suggests that Ca(2+) ions required for cell elongation might be supplied from the vacuoles located in the cortical region of the cell instead of Ca(2+) influx from the surrounding medium.

  14. Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light.

    PubMed

    Ma, Dingbang; Li, Xu; Guo, Yongxia; Chu, Jingfang; Fang, Shuang; Yan, Cunyu; Noel, Joseph P; Liu, Hongtao

    2016-01-01

    Cryptochrome 1 (CRY1) is a blue light receptor that mediates primarily blue-light inhibition of hypocotyl elongation. Very little is known of the mechanisms by which CRY1 affects growth. Blue light and temperature are two key environmental signals that profoundly affect plant growth and development, but how these two abiotic factors integrate remains largely unknown. Here, we show that blue light represses high temperature-mediated hypocotyl elongation via CRY1. Furthermore, CRY1 interacts directly with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) in a blue light-dependent manner to repress the transcription activity of PIF4. CRY1 represses auxin biosynthesis in response to elevated temperature through PIF4. Our results indicate that CRY1 signal by modulating PIF4 activity, and that multiple plant photoreceptors [CRY1 and PHYTOCHROME B (PHYB)] and ambient temperature can mediate morphological responses through the same signaling component-PIF4.

  15. Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light

    PubMed Central

    Ma, Dingbang; Li, Xu; Guo, Yongxia; Chu, Jingfang; Fang, Shuang; Yan, Cunyu; Noel, Joseph P.; Liu, Hongtao

    2016-01-01

    Cryptochrome 1 (CRY1) is a blue light receptor that mediates primarily blue-light inhibition of hypocotyl elongation. Very little is known of the mechanisms by which CRY1 affects growth. Blue light and temperature are two key environmental signals that profoundly affect plant growth and development, but how these two abiotic factors integrate remains largely unknown. Here, we show that blue light represses high temperature-mediated hypocotyl elongation via CRY1. Furthermore, CRY1 interacts directly with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) in a blue light-dependent manner to repress the transcription activity of PIF4. CRY1 represses auxin biosynthesis in response to elevated temperature through PIF4. Our results indicate that CRY1 signal by modulating PIF4 activity, and that multiple plant photoreceptors [CRY1 and PHYTOCHROME B (PHYB)] and ambient temperature can mediate morphological responses through the same signaling component—PIF4. PMID:26699514

  16. Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light.

    PubMed

    Ma, Dingbang; Li, Xu; Guo, Yongxia; Chu, Jingfang; Fang, Shuang; Yan, Cunyu; Noel, Joseph P; Liu, Hongtao

    2016-01-01

    Cryptochrome 1 (CRY1) is a blue light receptor that mediates primarily blue-light inhibition of hypocotyl elongation. Very little is known of the mechanisms by which CRY1 affects growth. Blue light and temperature are two key environmental signals that profoundly affect plant growth and development, but how these two abiotic factors integrate remains largely unknown. Here, we show that blue light represses high temperature-mediated hypocotyl elongation via CRY1. Furthermore, CRY1 interacts directly with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) in a blue light-dependent manner to repress the transcription activity of PIF4. CRY1 represses auxin biosynthesis in response to elevated temperature through PIF4. Our results indicate that CRY1 signal by modulating PIF4 activity, and that multiple plant photoreceptors [CRY1 and PHYTOCHROME B (PHYB)] and ambient temperature can mediate morphological responses through the same signaling component-PIF4. PMID:26699514

  17. The elongation of yeast prion fibers involves separable steps of association and conversion

    PubMed Central

    Scheibel, Thomas; Bloom, Jesse; Lindquist, Susan L.

    2004-01-01

    A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion [PSI+], a protein-based genetic element of Saccharomyces cerevisiae. In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of nucleated conformational conversion. First, oligomeric species convert to nuclei, and these nuclei then promote polymerization of soluble protein into amyloid fibers. To elucidate the nature of the polymerization step, we created single-cysteine substitution mutants at different positions in NM to provide unique attachment sites for various probes. In vivo, the mutants behaved like wild-type protein in both the [psi–] and [PSI+] states. In vitro, they assembled with wild-type kinetics and formed fibers with the same morphologies. When labeled with fluorescent probes, two mutants, NMT158C and NME167C, exhibited a change in fluorescence coincident with amyloid assembly. These mutants provided a sensitive measure for the kinetics of fiber elongation, and the lag phase in conversion. The cysteine in the mutant NMK184C remained exposed after assembly. When labeled with biotin and bound to streptavidin beads, it was used to capture radiolabeled soluble NM in the process of conversion. This process established the existence of a detergent-susceptible intermediate in fiber elongation. Thus, the second stage of nucleated conformational conversion, fiber elongation, itself contains at least two steps: the association of soluble protein with preformed fibers to form an assembly intermediate, followed by conformational conversion into amyloid. PMID:14983002

  18. Gibberellin-Regulation and Genetic Variations in Leaf Elongation for Tall Fescue in Association with Differential Gene Expression Controlling Cell Expansion

    PubMed Central

    Xu, Qian; Krishnan, Sanalkumar; Merewitz, Emily; Xu, Jichen; Huang, Bingru

    2016-01-01

    Leaf elongation rate (LER) is an important factor controlling plant growth and productivity. The objective of this study was to determine whether genetic variation in LER for a fast-growing (‘K-31’), and a dwarf cultivar (‘Bonsai’) of tall fescue (Festuca arundinacea) and gibberellic acid (GA) regulation of LER were associated with differential expression of cell-expansion genes. Plants were treated with GA3, trinexapac-ethyl (TE) (GA inhibitor), or water (untreated control) in a hydroponic system. LER of ‘K-31’ was 63% greater than that of ‘Bonsai’, which corresponded with 32% higher endogenous GA4 content in leaf and greater cell elongation and production rates under the untreated control condition. Exogenous application of GA3 significantly enhanced LER while TE treatment inhibited leaf elongation due to GA3-stimulation or TE-inhibition of cell elongation and production rate in leaves for both cultivars. Real-time quantitative polymerase chain reaction analysis revealed that three α-expansins, one β-expansin, and three xyloglucan endotransglycosylase (XET) genes were associated with GA-stimulation of leaf elongation, of which, the differential expression of EXPA4 and EXPA7 was related to the genotypic variation in LER of two cultivars. Those differentially-expressed expansin and XET genes could play major roles in genetic variation and GA-regulated leaf elongation in tall fescue. PMID:27457585

  19. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation.

    PubMed

    Vallerga, María Belén; Mansilla, Sabrina F; Federico, María Belén; Bertolin, Agustina P; Gottifredi, Vanesa

    2015-12-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.

  20. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

    PubMed Central

    Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

    2015-01-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

  1. Gibberellin-Regulation and Genetic Variations in Leaf Elongation for Tall Fescue in Association with Differential Gene Expression Controlling Cell Expansion.

    PubMed

    Xu, Qian; Krishnan, Sanalkumar; Merewitz, Emily; Xu, Jichen; Huang, Bingru

    2016-01-01

    Leaf elongation rate (LER) is an important factor controlling plant growth and productivity. The objective of this study was to determine whether genetic variation in LER for a fast-growing ('K-31'), and a dwarf cultivar ('Bonsai') of tall fescue (Festuca arundinacea) and gibberellic acid (GA) regulation of LER were associated with differential expression of cell-expansion genes. Plants were treated with GA3, trinexapac-ethyl (TE) (GA inhibitor), or water (untreated control) in a hydroponic system. LER of 'K-31' was 63% greater than that of 'Bonsai', which corresponded with 32% higher endogenous GA4 content in leaf and greater cell elongation and production rates under the untreated control condition. Exogenous application of GA3 significantly enhanced LER while TE treatment inhibited leaf elongation due to GA3-stimulation or TE-inhibition of cell elongation and production rate in leaves for both cultivars. Real-time quantitative polymerase chain reaction analysis revealed that three α-expansins, one β-expansin, and three xyloglucan endotransglycosylase (XET) genes were associated with GA-stimulation of leaf elongation, of which, the differential expression of EXPA4 and EXPA7 was related to the genotypic variation in LER of two cultivars. Those differentially-expressed expansin and XET genes could play major roles in genetic variation and GA-regulated leaf elongation in tall fescue. PMID:27457585

  2. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation.

    PubMed

    Vallerga, María Belén; Mansilla, Sabrina F; Federico, María Belén; Bertolin, Agustina P; Gottifredi, Vanesa

    2015-12-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

  3. Transcription elongation and tissue-specific somatic CAG instability.

    PubMed

    Goula, Agathi-Vasiliki; Stys, Agnieszka; Chan, Jackson P K; Trottier, Yvon; Festenstein, Richard; Merienne, Karine

    2012-01-01

    The expansion of CAG/CTG repeats is responsible for many diseases, including Huntington's disease (HD) and myotonic dystrophy 1. CAG/CTG expansions are unstable in selective somatic tissues, which accelerates disease progression. The mechanisms underlying repeat instability are complex, and it remains unclear whether chromatin structure and/or transcription contribute to somatic CAG/CTG instability in vivo. To address these issues, we investigated the relationship between CAG instability, chromatin structure, and transcription at the HD locus using the R6/1 and R6/2 HD transgenic mouse lines. These mice express a similar transgene, albeit integrated at a different site, and recapitulate HD tissue-specific instability. We show that instability rates are increased in R6/2 tissues as compared to R6/1 matched-samples. High transgene expression levels and chromatin accessibility correlated with the increased CAG instability of R6/2 mice. Transgene mRNA and H3K4 trimethylation at the HD locus were increased, whereas H3K9 dimethylation was reduced in R6/2 tissues relative to R6/1 matched-tissues. However, the levels of transgene expression and these specific histone marks were similar in the striatum and cerebellum, two tissues showing very different CAG instability levels, irrespective of mouse line. Interestingly, the levels of elongating RNA Pol II at the HD locus, but not the initiating form of RNA Pol II, were tissue-specific and correlated with CAG instability levels. Similarly, H3K36 trimethylation, a mark associated with transcription elongation, was specifically increased at the HD locus in the striatum and not in the cerebellum. Together, our data support the view that transcription modulates somatic CAG instability in vivo. More specifically, our results suggest for the first time that transcription elongation is regulated in a tissue-dependent manner, contributing to tissue-selective CAG instability. PMID:23209427

  4. Amorphous Zr-Based Foams with Aligned, Elongated Pores

    NASA Astrophysics Data System (ADS)

    Cox, Marie E.; Mathaudhu, Suveen N.; Hartwig, K. Ted; Dunand, David C.

    2010-07-01

    Interpenetrating phase composites are created by warm equal channel angular extrusion (ECAE) of blended powders of amorphous Zr58.5Nb2.8Cu15.6Ni12.8Al10.3 (Vit106a) and a crystalline ductile metal (Cu, Ni, or W). Subsequent dissolution of the continuous metallic phase results in amorphous Vit106a foams with ~40 pct aligned, elongated pores. The extent of Vit106a powder densification in the composites improves with the strength of the crystalline metallic powder, from low for Cu to high for W, with a concomitant improvement in foam compressive strength, ductility, and energy absorption.

  5. Elongated optical bottle beams generated by composite binary axicons

    NASA Astrophysics Data System (ADS)

    Porfirev, A. P.; Skidanov, R. V.

    2016-04-01

    We provide analytical, numerical and experimental study of the possibility of forming elongated optical bottle beams (OBBs) using composite binary phase axicons. In this case, the OBB is generated by the superposition of Bessel beams in the near-field region on the axicon. To generate the OBB experimentally, we utilized a spatial light modulator. The experimental results are qualitatively consistent with the results of numerical simulations performed using Fresnel transform. Such type of optical trap can be applied in many applications of microbiology, micromechanics and meteorology to manipulate micro- and nanoobjects in liquid or gaseous medium.

  6. Controlled laser production of elongated articles from particulates

    DOEpatents

    Dixon, Raymond D.; Lewis, Gary K.; Milewski, John O.

    2002-01-01

    It has been discovered that wires and small diameter rods can be produced using laser deposition technology in a novel way. An elongated article such as a wire or rod is constructed by melting and depositing particulate material into a deposition zone which has been designed to yield the desired article shape and dimensions. The article is withdrawn from the deposition zone as it is formed, thus enabling formation of the article in a continuous process. Alternatively, the deposition zone is moved along any of numerous deposition paths away from the article being formed.

  7. Reduction of laser spot elongation in adaptive optics.

    PubMed

    Ribak, Erez N; Ragazzoni, Roberto

    2004-06-15

    Adaptive optics systems measure the wave front to be corrected by use of a reference source, a star, or a laser beacon. Such laser guide stars are a few kilometers long, and when observed near the edges of large telescopes they appear elongated. This limits their utility significantly. However, with more sophisticated launch optics their shape and length can be controlled. We propose to string around the rim of a telescope a number of small telescopes that will add laser beams in the scattering medium to create a compact spot. The method could also be adapted for ocular adaptive optics.

  8. “Co-transcriptionality” - the transcription elongation complex as a nexus for nuclear transactions

    PubMed Central

    Perales, Roberto; Bentley, David

    2009-01-01

    Much of the complex process of RNP biogenesis takes place at the gene, co-transcriptionally. The target for RNA binding and processing factors is therefore not a solitary RNA molecule, but a transcription elongation complex (TEC) comprising the growing nascent RNA and RNA polymerase traversing a chromatin template with associated passenger proteins. RNA maturation factors are not the only nuclear machines whose work is organized co-transcriptionally around the TEC scaffold. In addition DNA repair, covalent chromatin modification, “gene gating” at the nuclear pore, Ig gene hypermutation, and sister chromosome cohesion have all been demonstrated or suggested to involve a co-transcriptional component. From this perspective, TEC’s can be viewed as potent “community organizers” within the nucleus. PMID:19854129

  9. Highly conserved base A55 of 16S ribosomal RNA is important for the elongation cycle of protein synthesis.

    PubMed

    Sahu, Bhubanananda; Khade, Prashant K; Joseph, Simpson

    2013-09-24

    Accurate decoding of mRNA requires the precise interaction of protein factors and tRNAs with the ribosome. X-ray crystallography and cryo-electron microscopy have provided detailed structural information about the 70S ribosome with protein factors and tRNAs trapped during translation. Crystal structures showed that one of the universally conserved 16S rRNA bases, A55, in the shoulder domain of the 30S subunit interacts with elongation factors Tu and G (EF-Tu and EF-G, respectively). The exact functional role of A55 in protein synthesis is not clear. We changed A55 to U and analyzed the effect of the mutation on the elongation cycle of protein synthesis using functional assays. Expression of 16S rRNA with the A55U mutation in cells confers a dominant lethal phenotype. Additionally, ribosomes with the A55U mutation in 16S rRNA show substantially reduced in vitro protein synthesis activity. Equilibrium binding studies showed that the A55U mutation considerably inhibited the binding of the EF-Tu·GTP·tRNA ternary complex to the ribosome. Furthermore, the A55U mutation slightly inhibited the peptidyl transferase reaction, the binding of EF-G·GTP to the ribosome, and mRNA-tRNA translocation. These results indicate that A55 is important for fine-tuning the activity of the ribosome during the elongation cycle of protein synthesis.

  10. Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment.

    PubMed

    Blasiak, Agata; Lee, Gil U; Kilinc, Devrim

    2015-09-16

    Correct wiring of the nervous system requires guidance cues, diffusible or substrate-bound proteins that steer elongating axons to their target tissues. Netrin-1, the best characterized member of the Netrins family of guidance molecules, is known to induce axon turning and modulate axon elongation rate; however, the factors regulating the axonal response to Netrin-1 are not fully understood. Using microfluidics, we treated fluidically isolated axons of mouse primary cortical neurons with Netrin-1 and characterized axon elongation rates, as well as the membrane localization of deleted in colorectal cancer (DCC), a well-established receptor of Netrin-1. The capacity to stimulate and observe a large number of individual axons allowed us to conduct distribution analyses, through which we identified two distinct neuron subpopulations based on different elongation behavior and different DCC membrane dynamics. Netrin-1 reduced the elongation rates in both subpopulations, where the effect was more pronounced in the slow growing subpopulation. Both the source of Ca(2+) influx and the basal cytosolic Ca(2+) levels regulated the effect of Netrin-1, for example, Ca(2+) efflux from the endoplasmic reticulum due to the activation of Ryanodine channels blocked Netrin-1-induced axon slowdown. Netrin-1 treatment resulted in a rapid membrane insertion of DCC, followed by a gradual internalization. DCC membrane dynamics were different in the central regions of the growth cones compared to filopodia and axon shafts, highlighting the temporal and spatial heterogeneity in the signaling events downstream of Netrin-1. Cumulatively, these results demonstrate the power of microfluidic compartmentalization and distribution analysis in describing the complex axonal Netrin-1 response. PMID:26190161

  11. Modeling cell elongation during germ band retraction: cell autonomy versus applied anisotropic stress

    NASA Astrophysics Data System (ADS)

    Lynch, Holley E.; Veldhuis, Jim; Brodland, G. Wayne; Hutson, M. Shane

    2014-05-01

    The morphogenetic process of germ band retraction in Drosophila embryos involves coordinated movements of two epithelial tissues—germ band and amnioserosa. The germ band shortens along its rostral-caudal or head-to-tail axis, widens along its perpendicular dorsal-ventral axis, and uncurls from an initial ‘U’ shape. The amnioserosa mechanically assists this process by pulling on the crook of the U-shaped germ band. The amnioserosa may also provide biochemical signals that drive germ band cells to change shape in a mechanically autonomous fashion. Here, we use a finite-element model to investigate how these two contributions reshape the germ band. We do so by modeling the response to laser-induced wounds in each of the germ band’s spatially distinct segments (T1-T3, A1-A9) during the middle of retraction when segments T1-A3 form the ventral arm of the ‘U’, A4-A7 form its crook, and A8-A9 complete the dorsal arm. We explore these responses under a range of externally applied stresses and internal anisotropy of cell edge tensions—akin to a planar cell polarity that can drive elongation of cells in a direction parallel to the minimum edge tension—and identify regions of parameter space (edge-tension anisotropy versus stress anisotropy) that best match previous experiments for each germ band segment. All but three germ band segments are best fit when the applied stress anisotropy and the edge-tension anisotropy work against one another—i.e., when the isolated effects would elongate cells in perpendicular directions. Segments in the crook of the germ band (A4-A7) have cells that elongate in the direction of maximum external stress, i.e., external stress anisotropy is dominant. In most other segments, the dominant factor is internal edge-tension anisotropy. These results are consistent with models in which the amnioserosa pulls on the crook of the germ band to mechanically assist retraction. In addition, they suggest a mechanical cue for edge

  12. Modeling cell elongation during germ band retraction: cell autonomy versus applied anisotropic stress

    PubMed Central

    Lynch, Holley E.; Veldhuis, Jim; Brodland, G. Wayne; Hutson, M. Shane

    2014-01-01

    The morphogenetic process of germ band retraction in Drosophila embryos involves coordinated movements of two epithelial tissues – germ band and amnioserosa. The germ band shortens along its rostral-caudal or head-to-tail axis, widens along its perpendicular dorsal-ventral axis, and uncurls from an initial ‘U’ shape. The amnioserosa mechanically assists this process by pulling on the crook of the U-shaped germ band. The amnioserosa may also provide biochemical signals that drive germ band cells to change shape in a mechanically autonomous fashion. Here, we use a finite-element model to investigate how these two contributions reshape the germ band. We do so by modeling the response to laser-induced wounds in each of the germ band’s spatially distinct segments (T1-T3, A1-A9) during the middle of retraction when segments T1-A3 form the ventral arm of the ‘U’, A4-A7 form its crook, and A8-A9 complete the dorsal arm. We explore these responses under a range of externally applied stresses and internal anisotropy of cell edge tensions – akin to a planar cell polarity that can drive elongation of cells in a direction parallel to the minimum edge tension – and identify regions of parameter space (edge-tension anisotropy versus stress anisotropy) that best match previous experiments for each germ band segment. All but three germ band segments are best fit when the applied stress anisotropy and the edge-tension anisotropy work against one another – i.e., when the isolated effects would elongate cells in perpendicular directions. Segments in the crook of the germ band (A4-A7) have cells that elongate in the direction of maximum external stress, i.e., external stress anisotropy is dominant. In most other segments, the dominant factor is internal edge-tension anisotropy. These results are consistent with models in which the amnioserosa pulls on the crook of the germ band to mechanically assist retraction. In addition, they suggest a mechanical cue for edge

  13. The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.

    PubMed

    Leprivier, Gabriel; Remke, Marc; Rotblat, Barak; Dubuc, Adrian; Mateo, Abigail-Rachele F; Kool, Marcel; Agnihotri, Sameer; El-Naggar, Amal; Yu, Bin; Somasekharan, Syam Prakash; Faubert, Brandon; Bridon, Gaëlle; Tognon, Cristina E; Mathers, Joan; Thomas, Ryan; Li, Amy; Barokas, Adi; Kwok, Brian; Bowden, Mary; Smith, Stephanie; Wu, Xiaochong; Korshunov, Andrey; Hielscher, Thomas; Northcott, Paul A; Galpin, Jason D; Ahern, Christopher A; Wang, Ye; McCabe, Martin G; Collins, V Peter; Jones, Russell G; Pollak, Michael; Delattre, Olivier; Gleave, Martin E; Jan, Eric; Pfister, Stefan M; Proud, Christopher G; Derry, W Brent; Taylor, Michael D; Sorensen, Poul H

    2013-05-23

    Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

  14. Fluoride exposure regulates the elongation phase of protein synthesis in cultured Bergmann glia cells.

    PubMed

    Flores-Méndez, Marco; Ramírez, Diana; Alamillo, Nely; Hernández-Kelly, Luisa C; Del Razo, Luz María; Ortega, Arturo

    2014-08-17

    Fluoride is an environmental pollutant present in dental products, food, pesticides and water. The latter, is the greatest source of exposure to this contaminant. Structural and functional damages to the central nervous system are present in exposed population. An established consequence of the neuronal is the release of a substantial amount of glutamate to the extracellular space, leading to an excitotoxic insult. Glutamate exerts its actions through the activation of specific plasma membrane receptors and transporters present in neurons and in glia cells and it is the over-activation of glutamate receptors and transporters, the biochemical hallmark of neuronal and oligodendrocyte cell death. In this context, taking into consideration that fluoride leads to degeneration of cerebellar cells, we took the advantage of the well-established model of cerebellar Bergmann glia cultures to gain insight into the molecular mechanisms inherent to fluoride neurotoxicity that might be triggered in glia cells. We could establish that fluoride decreases [(35)S]-methionine incorporation into newly synthesized polypeptides, in a time-dependent manner, and that this halt in protein synthesis is the result of a decrease in the elongation phase of translation, mediated by an augmentation of eukaryotic elongation factor 2 phosphorylation. These results favor the notion of glial cells as targets of fluoride toxicity and strengthen the idea of a critical involvement of glia cells in the function and dysfunction of the brain. PMID:24954634

  15. Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation

    PubMed Central

    Bertucci, Paola Y.; Nacht, A. Silvina; Alló, Mariano; Rocha-Viegas, Luciana; Ballaré, Cecilia; Soronellas, Daniel; Castellano, Giancarlo; Zaurin, Roser; Kornblihtt, Alberto R.; Beato, Miguel; Vicent, Guillermo P.; Pecci, Adali

    2013-01-01

    Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3′-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes. PMID:23640331

  16. Proton and calcium flux oscillations in the elongation region correlate with root nutation.

    PubMed

    Shabala, S N; Newman, I A

    1997-08-01

    The involvement of Ca2+ and H+ flux oscillations in root nutation was studied for decapped roots of corn (Zea mays L. cv. Aussie Gold) placed horizontally. Net ion fluxes were measured around the elongation and meristematic regions using a microelectrode ion flux measuring system. High correlation between H+ flux oscillations and root nutations was found in the elongation region. Two oscillatory components of H+ flux, with periods of about 90 min and 7 min, correlated with root circumnutations and micronutations, respectively. The periods of H+ flux oscillations and rhythmical root movements in this region could be modified similarly by external factors including pH. In the meristematic region no association between ion flux behaviour and nutation was apparent. Ion flux oscillations and nutations both decreased in amplitude as the growth rate at the measured location decreased. Possible involvement of ion flux oscillations in root circumnutation is discussed. It is concluded that a model involving an internal oscillator must be developed to explain the H+ flux involvement in root nutations. PMID:11540486

  17. Expansin-regulated cell elongation is involved in the drought tolerance in wheat.

    PubMed

    Zhao, Mei-rong; Li, Feng; Fang, Ying; Gao, Qiang; Wang, Wei

    2011-04-01

    Water stress restrains plant growth. Expansin is a cell wall protein that is generally accepted to be the key regulator of cell wall extension during plant growth. In this study, we used two different wheat cultivars to study the involvement of expansin in drought tolerance. Wheat coleoptile was used as the material in experiment. Our results indicated that water stress induced an increase in acidic pH-dependant cell wall extension, which is related to expansin activity; however, water stress inhibited coleoptile elongation growth. The increased expansin activity was mainly due to increased expression of expansin protein that was upregulated by water stress, but water stress also resulted in a decrease in cell wall acidity, a negative factor for cell wall extension. Decreased plasma membrane H(+)-ATPase activity was involved in the alkalinization of the cell wall under water stress. The activity of expansin in HF9703 (a drought-tolerant wheat cultivar) was always higher than that in 921842 (a drought-sensitive wheat cultivar) under both normal and water stress conditions, which may be correlated with the higher expansin protein expression and plasma membrane H(+)-ATPase activity observed in HF9703 versus 921842. However, water stress did not change the susceptibility of the wheat cell wall to expansin, and no difference in this susceptibility was observed between the drought-tolerant and drought-sensitive wheat cultivars. These results suggest the involvement of expansin in cell elongation and the drought resistance of wheat.

  18. Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells.

    PubMed

    Gazave, Eve; Béhague, Julien; Laplane, Lucie; Guillou, Aurélien; Préau, Laetitia; Demilly, Adrien; Balavoine, Guillaume; Vervoort, Michel

    2013-10-01

    Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the "segment addition zone" (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral "stemness" program. PMID:23891818

  19. The eEF2 Kinase Confers Resistance to Nutrient Deprivation by Blocking Translation Elongation

    PubMed Central

    Leprivier, Gabriel; Remke, Marc; Rotblat, Barak; Dubuc, Adrian; Mateo, Abigail-Rachele F.; Kool, Marcel; Agnihotri, Sameer; El-Naggar, Amal; Yu, Bin; Somasekharan, Syam Prakash; Faubert, Brandon; Bridon, Gaëlle; Tognon, Cristina E.; Mathers, Joan; Thomas, Ryan; Li, Amy; Barokas, Adi; Kwok, Brian; Bowden, Mary; Smith, Stephanie; Wu, Xiaochong; Korshunov, Andrey; Hielscher, Thomas; Northcott, Paul A.; Galpin, Jason D.; Ahern, Christopher A.; Wang, Ye; McCabe, Martin G.; Collins, V. Peter; Jones, Russell G.; Pollak, Michael; Delattre, Olivier; Gleave, Martin E.; Jan, Eric; Pfister, Stefan M.; Proud, Christopher G.; Derry, W. Brent; Taylor, Michael D.; Sorensen, Poul H.

    2015-01-01

    SUMMARY Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PMID:23706743

  20. Crystal structure of the three tandem FF domains of the transcription elongation regulator CA150.

    PubMed

    Lu, Ming; Yang, Jun; Ren, Zhiyong; Sabui, Subir; Espejo, Alexsandra; Bedford, Mark T; Jacobson, Raymond H; Jeruzalmi, David; McMurray, John S; Chen, Xiaomin

    2009-10-23

    FF domains are small protein-protein interaction modules that have two flanking conserved phenylalanine residues. They are present in proteins involved in transcription, RNA splicing, and signal transduction, and often exist in tandem arrays. Although several individual FF domain structures have been determined by NMR, the tandem nature of most FF domains has not been revealed. Here we report the 2.7-A-resolution crystal structure of the first three FF domains of the human transcription elongation factor CA150. Each FF domain is composed of three alpha-helices and a 3(10) helix between alpha-helix 2 and alpha-helix 3. The most striking feature of the structure is that an FF domain is connected to the next by an alpha-helix that continues from helix 3 to helix 1 of the next. The consequent elongated arrangement allows exposure of many charged residues within the region that can be engaged in interaction with other molecules. Binding studies using a peptide ligand suggest that a specific conformation of the FF domains might be required to achieve higher-affinity binding. Additionally, we explore potential DNA binding of the FF construct used in this study. Overall, we provide the first crystal structure of an FF domain and insights into the tandem nature of the FF domains and suggest that, in addition to protein binding, FF domains might be involved in DNA binding.

  1. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

    PubMed

    Min, Irene M; Waterfall, Joshua J; Core, Leighton J; Munroe, Robert J; Schimenti, John; Lis, John T

    2011-04-01

    Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

  2. Chromosome end elongation by recombination in the mosquito Anopheles gambiae.

    PubMed Central

    Roth, C W; Kobeski, F; Walter, M F; Biessmann, H

    1997-01-01

    One of the functions of telomeres is to counteract the terminal nucleotide loss associated with DNA replication. While the vast majority of eukaryotic organisms maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, other mechanisms such as the transposition of retrotransposons or recombination can also be used in some species. Chromosome end regression and extension were studied in a medically important mosquito, the malaria vector Anopheles gambiae, to determine how this dipteran insect maintains its chromosome ends. The insertion of a transgenic pUChsneo plasmid at the left end of chromosome 2 provided a unique marker for measuring the dynamics of the 2L telomere over a period of about 3 years. The terminal length was relatively uniform in the 1993 population with the chromosomes ending within the white gene sequence of the inserted transgene. Cloned terminal chromosome fragments did not end in short repeat sequences that could have been synthesized by telomerase. By late 1995, the chromosome ends had become heterogeneous: some had further shortened while other chromosomes had been elongated by regenerating part of the integrated pUChsneo plasmid. A model is presented for extension of the 2L chromosome by recombination between homologous 2L chromosome ends by using the partial plasmid duplication generated during its original integration. It is postulated that this mechanism is also important in wild-type telomere elongation. PMID:9271395

  3. The Brassica rapa elongated internode (EIN) gene encodes phytochrome B.

    PubMed

    Devlin, P F; Somers, D E; Quail, P H; Whitelam, G C

    1997-06-01

    The elongated internode (ein) mutation of Brassica rapa leads to a deficiency in immunochemically detectable phytochrome B. Molecular analysis of the PHYB gene from ein indicates a deletion in the flanking DNA 5' of the ATG start codon, which could interfere either with PHYB transcription or processing of the PHYB transcript. Restriction fragment length polymorphisms and inverse PCR fragments generated from the PHYB gene of wild-type and ein seedlings demonstrate the deletion to be 500 bp in length. Seedlings of heterozygote, EIN/ein, contain about 50% of the level of immunochemically detectable phytochrome B of equivalent wild-type EIN/EIN seedlings. Etiolated seedlings of EIN/ein show a responsiveness to red light almost intermediate between that of ein/ein and EIN/EIN homozygotes. Furthermore, whereas the ein/ein homozygote is poorly responsive to low red/far-red ratio light, the presence of one functional allele of EIN in the heterozygote confers an elongation response intermediate between that of the homozygotes EIN/EIN and ein/ein in these light conditions. The partial dominance of ein indicates a close relationship between phytochrome B level and phenotype.

  4. Electrostatics in the Ribosomal Tunnel Modulate Chain Elongation Rates

    PubMed Central

    Lu, Jianli; Deutsch, Carol

    2008-01-01

    SUMMARY Electrostatic potentials along the ribosomal exit tunnel are non-uniform and negative. The significance of electrostatics in the tunnel remains relatively uninvestigated, yet is likely to play a role in translation and secondary folding of nascent peptides. To probe the role of nascent peptide charges in ribosome function, we used a molecular tape measure that was engineered to contain different numbers of charged amino acids localized to known regions of the tunnel, and measured chain elongation rates. Positively-charged arginine or lysine sequences produce transient arrest (pausing) before the nascent peptide is fully elongated. The rate of conversion from transiently arrested to full-length nascent peptide is faster for peptides containing neutral or negatively-charged residues than for those containing positively-charged residues. We provide experimental evidence that extra-ribosomal mechanisms do not account for this charge-specific pausing. We conclude that pausing is due to charge-specific interactions between the tunnel and the nascent peptide. PMID:18822297

  5. Application of an Elongated Kelvin Model to Space Shuttle Foams

    NASA Technical Reports Server (NTRS)

    Sullivan, Roy M.; Ghosn, Louis J.; Lerch, Bradley A.

    2009-01-01

    The space shuttle foams are rigid closed-cell polyurethane foams. The two foams used most-extensively oil space shuttle external tank are BX-265 and NCFL4-124. Because of the foaming and rising process, the foam microstructures are elongated in the rise direction. As a result, these two foams exhibit a nonisotropic mechanical behavior. A detailed microstructural characterization of the two foams is presented. Key features of the foam cells are described and the average cell dimensions in the two foams are summarized. Experimental studies are also conducted to measure the room temperature mechanical response of the two foams in the two principal material directions (parallel to the rise and perpendicular to the rise). The measured elastic modulus, proportional limit stress, ultimate tensile strength, and Poisson's ratios are reported. The generalized elongated Kelvin foam model previously developed by the authors is reviewed and the equations which result from this model are summarized. Using the measured microstructural dimensions and the measured stiffness ratio, the foam tensile strength ratio and Poisson's ratios are predicted for both foams and are compared with the experimental data. The predicted tensile strength ratio is in close agreement with the measured strength ratio for both BX-265 and NCFI24-124. The comparison between the predicted Poisson's ratios and the measured values is not as favorable.

  6. Directed 3D cell alignment and elongation in microengineered hydrogels.

    PubMed

    Aubin, Hug; Nichol, Jason W; Hutson, Ché B; Bae, Hojae; Sieminski, Alisha L; Cropek, Donald M; Akhyari, Payam; Khademhosseini, Ali

    2010-09-01

    Organized cellular alignment is critical to controlling tissue microarchitecture and biological function. Although a multitude of techniques have been described to control cellular alignment in 2D, recapitulating the cellular alignment of highly organized native tissues in 3D engineered tissues remains a challenge. While cellular alignment in engineered tissues can be induced through the use of external physical stimuli, there are few simple techniques for microscale control of cell behavior that are largely cell-driven. In this study we present a simple and direct method to control the alignment and elongation of fibroblasts, myoblasts, endothelial cells and cardiac stem cells encapsulated in microengineered 3D gelatin methacrylate (GelMA) hydrogels, demonstrating that cells with the intrinsic potential to form aligned tissues in vivo will self-organize into functional tissues in vitro if confined in the appropriate 3D microarchitecture. The presented system may be used as an in vitro model for investigating cell and tissue morphogenesis in 3D, as well as for creating tissue constructs with microscale control of 3D cellular alignment and elongation, that could have great potential for the engineering of functional tissues with aligned cells and anisotropic function.

  7. Local auxin metabolism regulates environment-induced hypocotyl elongation.

    PubMed

    Zheng, Zuyu; Guo, Yongxia; Novák, Ondřej; Chen, William; Ljung, Karin; Noel, Joseph P; Chory, Joanne

    2016-01-01

    A hallmark of plants is their adaptability of size and form in response to widely fluctuating environments. The metabolism and redistribution of the phytohormone auxin play pivotal roles in establishing active auxin gradients and resulting cellular differentiation. In Arabidopsis thaliana, cotyledons and leaves synthesize indole-3-acetic acid (IAA) from tryptophan through indole-3-pyruvic acid (3-IPA) in response to vegetational shade. This newly synthesized auxin moves to the hypocotyl where it induces elongation of hypocotyl cells. Here we show that loss of function of VAS2 (IAA-amido synthetase Gretchen Hagen 3 (GH3).17) leads to increases in free IAA at the expense of IAA-Glu (IAA-glutamate) in the hypocotyl epidermis. This active IAA elicits shade- and high temperature-induced hypocotyl elongation largely independently of 3-IPA-mediated IAA biosynthesis in cotyledons. Our results reveal an unexpected capacity of local auxin metabolism to modulate the homeostasis and spatial distribution of free auxin in specialized organs such as hypocotyls in response to shade and high temperature. PMID:27249562

  8. Initiation and elongation of lateral roots in Lactuca sativa

    NASA Technical Reports Server (NTRS)

    Zhang, N.; Hasenstein, K. H.

    1999-01-01

    Lactuca sativa cv. Baijianye seedlings do not normally produce lateral roots, but removal of the root tip or application of auxin, especially indole-butyric acid, triggered the formation of lateral roots. Primordia initiated within 9 h and were fully developed after 24 h by activating the pericycle cells opposite the xylem pole. The pericycle cells divided asymmetrically into short and long cells. The short cells divided further to form primordia. The effect of root tip removal and auxin application was reversed by 6-benzylaminopurine at concentrations >10(-8) M. The cytokinin oxidase inhibitor N1-(2chloro4pyridyl)-N2-phenylurea also suppressed auxin-induced lateral rooting. The elongation of primary roots was promoted by L-alpha-(2-aminoethoxyvinyl) glycine and silver ions, but only the latter enhanced elongation of lateral roots. The data indicate that the induction of lateral roots is controlled by basipetally moving cytokinin and acropetally moving auxin. Lateral roots appear to not produce ethylene.

  9. Local auxin metabolism regulates environment-induced hypocotyl elongation

    PubMed Central

    Zheng, Zuyu; Guo, Yongxia; Novák, Ondřej; Chen, William; Ljung, Karin; Noel, Joseph P.; Chory, Joanne

    2016-01-01

    A hallmark of plants is their adaptability of size and form in response to widely fluctuating environments. The metabolism and redistribution of the phytohormone auxin play pivotal roles in establishing active auxin gradients and resulting cellular differentiation. In Arabidopsis thaliana, cotyledons and leaves synthesize indole-3-acetic acid (IAA) from tryptophan through indole-3-pyruvic acid (3-IPA) in response to vegetational shade. This newly synthesized auxin moves to the hypocotyl where it induces elongation of hypocotyl cells. Here we show that loss of function of VAS2 (IAA-amido synthetase Gretchen Hagen 3 (GH3).17) leads to increases in free IAA at the expense of IAA-Glu (IAA-glutamate) in the hypocotyl epidermis. This active IAA elicits shade- and high temperature-induced hypocotyl elongation largely independently of 3-IPA-mediated IAA biosynthesis in cotyledons. Our results reveal an unexpected capacity of local auxin metabolism to modulate the homeostasis and spatial distribution of free auxin in specialized organs such as hypocotyls in response to shade and high temperature. PMID:27249562

  10. Study of Elongated Superbubble around CYG OB1

    NASA Technical Reports Server (NTRS)

    Sonneborn, George (Technical Monitor); Nichols, Joy

    2004-01-01

    OB association superbubbles and supernova remnants are believed to be the primary sources of the hot component of the interstellar medium. Super- bubbles are also thought to "blow out" of the galactic plane and are thus the potential source of galactic coronal gas. Quantifying the energetics and accurately modeling the evolution of superbubbles is thus an important task in the understanding of the evolution of our Galaxy. The superbubble surrounding the Cyg OB1 association is an excellent example for study of the early stages of superbubble evolution. This superbubble, discovered in IRAS data, is an elongated structure 2 x 5 degrees (26 x 65 pc at distance=1.5 kpc), with at least 2 known supernova remnants believed to be associated with the superbubble. The elongated shape is more extended than can be explained by differential galactic rotation. A major problem in understanding the evolution of the Cyg OB1 superbubble is the discrepancy between the age of the superbubble and the derived age of the parent star association. The superbubble is believed to be no more than 1 Myr old, while the association is about 4-5 Myr old.

  11. Device for measuring hole elongation in a bolted joint

    NASA Technical Reports Server (NTRS)

    Wichorek, Gregory R. (Inventor)

    1987-01-01

    A device to determine the operable failure mode of mechanically fastened lightweight composite joints by measuring the hole elongation of a bolted joint is disclosed. The double-lap joint test apparatus comprises a stud, a test specimen having a hole, two load transfer plates, and linear displacement measuring instruments. The test specimen is sandwiched between the two load transfer plates and clamped together with the stud. Spacer washers are placed between the test specimen and each load transfer plate to provide a known, controllable area for the determination of clamping forces around the hole of the specimen attributable to bolt torque. The spacer washers also provide a gap for the mounting of reference angles on each side of the test specimen. Under tensile loading, elongation of the hole of the test specimen causes the stud to move away from the reference angles. This displacement is measured by the voltage output of two linear displacement measuring instruments that are attached to the stud and remain in contact with the reference angles throughout the tensile loading. The present invention obviates previous problems in obtaining specimen deformation measurements by monitoring the reference angles to the test specimen and the linear displacement measuring instruments to the stud.

  12. Elongation of discotic liquid crystal strands and lubricant effects.

    PubMed

    Bhattacharyya, Surjya Sarathi; Galerne, Yves

    2014-05-19

    After a short review on the physics of pulled threads and their mechanical properties, the paper reports and discusses the strand elongation of disordered columnar phases, hexagonal or lamella-columnar, of small molecules or polymers. The mechanical properties appear to be relevant to the length of the columns of molecules compared to the thread length, instead of the usual correlation length. If, taking the entanglement effect into account, the column length is short, the strand exhibits rather fluid-like properties that may even look nematic-like at the macroscopic scale. The Plateau-Rayleigh instability breaks the thread shortly thereafter. However, because the hydrodynamic objects are the columns instead of the molecules, the viscosity is anomalously large. The observations show that the strands in the columnar phases are made of filaments, or fibrils, which are bundles of columns of molecules. This explains the grooves and rings, which are observed on the antenna or bamboo-like strand profiles. On pulling a strand, the elongation stress eventually exceeds the plasticity threshold, thus breaking the columns and the filaments. As a result, cracks, more exactly, giant dislocations are formed. These change the strand thickness by steps of different birefringence colors. Interestingly, the addition of a solute may drastically change the effective viscosity of the columnar phase and its mechanical properties. Some solutes, such as alkanes, exhibit lubricant and detangling properties, whereas others such as triphenylene, are antilubricant. PMID:24302445

  13. Assessment of Spatial Distribution of Growth in the Elongation Zone of Grass Leaf Blades 1

    PubMed Central

    Schnyder, Hans; Nelson, Curtis J.; Coutts, John H.

    1987-01-01

    Knowledge about the spatial distribution of growth is essential for understanding the leaf growth process. In grasses the elongation zone is located at the base of the leaf blade and is enclosed by sheaths of older leaves. Assessment of spatial growth distribution, therefore, necessitates use of a destructive method. We used a fine needle to make holes through bases of tillers at the location of the leaf elongation zone of tall fescue (Festuca arundinacea Schreb.), then measured the displacement of the holes after a 6 or 24 h interval. Needle holes caused a 22 to 41% decrease in daily leaf elongation so experiments were conducted to investigate if the spatial distribution of growth in the elongation zone was altered. Leaf elongation rate was reduced similarly when needle holes were made within or above the zone where cell elongation occurs. Distribution of elongation within the zone was the same when estimated by displacement of needle holes or ink marks placed on the epidermis of the elongation zone after surrounding tissue had been removed. Making holes at different locations within the elongation zone did not differentially affect the relative contribution of the damaged or undamaged parts to leaf elongation. These findings demonstrate that needle holes or ink marks in paired leaves can be used to estimate the relative distribution of growth in the elongation zone of undamaged tall fescue leaf blades. PMID:16665672

  14. Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice

    PubMed Central

    Ayano, Madoka; Kani, Takahiro; Kojima, Mikiko; Sakakibara, Hitoshi; Kitaoka, Takuya; Kuroha, Takeshi; Angeles-Shim, Rosalyn B; Kitano, Hidemi; Nagai, Keisuke; Ashikari, Motoyuki

    2014-01-01

    Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice. Deepwater rice obtained the ability for rapid internode elongation to avoid drowning and adapt to flooded condition. How does it regulate internode elongation? Using both physiological and genetic approach, this paper shows that the plant hormone, gibberellin (GA) regulates internode elongation. PMID:24891164

  15. 60S ribosomal protein L35 regulates β-casein translational elongation and secretion in bovine mammary epithelial cells.

    PubMed

    Jiang, Nan; Hu, Lijun; Liu, Chaonan; Gao, Xueli; Zheng, Shimin

    2015-10-01

    60S ribosomal protein L35 (RPL35) is an important component of the 60S ribosomal subunit and has a role in protein translation and endoplasmic reticulum (ER) docking. However, few studies have investigated RPL35 in eukaryotes and much remains to be learned. Here, we analyzed the function of RPL35 in β-casein (CSN2) synthesis and secretion in bovine mammary epithelial cells (BMECs). We found that methionine (Met) could promote the expressions of CSN2 and RPL35. Analysis of overexpression and inhibition of RPL35 confirmed that it could mediate the Met signal and regulate CSN2 expression. The mechanism of CSN2 regulation by RPL35 was analyzed by coimmunoprecipitation (Co-IP), colocalization, fluorescence resonance energy transfer (FRET) and gene mutation. We found that RPL35 could control ribosome translational elongation during synthesis of CSN2 by interacting with eukaryotic translational elongation factor 2 (eEF2), and that eEF2 was the signaling molecule downstream of RPL35 controlling this process. RPL35 could also control the secretion of CSN2 by locating it to the ER. Taken together, these results revealed that, RPL35 was an important positive regulatory factor involving in the Met-mediated regulation of CSN2 translational elongation and secretion.

  16. CLASH: THE ENHANCED LENSING EFFICIENCY OF THE HIGHLY ELONGATED MERGING CLUSTER MACS J0416.1-2403

    SciTech Connect

    Zitrin, A.; Bartelmann, M.; Carrasco, M.; Meneghetti, M.; Umetsu, K.; Broadhurst, T.; Bradley, L.; Coe, D.; Koekemoer, A. M.; Postman, M.; Ford, H.; Medezinski, E.; Kelson, D.; Moustakas, J.; Moustakas, L. A.; Nonino, M.; Rosati, P.; Seidel, G.; Seitz, S.; and others

    2013-01-10

    We perform a strong lensing analysis of the merging galaxy cluster MACS J0416.1-2403 (M0416; z = 0.42) in recent CLASH/HST observations. We identify 70 new multiple images and candidates of 23 background sources in the range 0.7 {approx}< z{sub phot} {approx}< 6.14 including two probable high-redshift dropouts, revealing a highly elongated lens with axis ratio {approx_equal}5:1, and a major axis of {approx}100'' (z{sub s} {approx} 2). Compared to other well-studied clusters, M0416 shows an enhanced lensing efficiency. Although the critical area is not particularly large ({approx_equal} 0.6 {open_square}'; z{sub s} {approx} 2), the number of multiple images, per critical area, is anomalously high. We calculate that the observed elongation boosts the number of multiple images, per critical area, by a factor of {approx}2.5 Multiplication-Sign , due to the increased ratio of the caustic area relative to the critical area. Additionally, we find that the observed separation between the two main mass components enlarges the critical area by a factor of {approx}2. These geometrical effects can account for the high number (density) of multiple images observed. We find in numerical simulations that only {approx}4% of the clusters (with M{sub vir} {>=} 6 Multiplication-Sign 10{sup 14} h {sup -1} M{sub Sun }) exhibit critical curves as elongated as in M0416.

  17. Gibberellin deficiency and response mutations suppress the stem elongation phenotype of phytochrome-deficient mutants of Arabidopsis.

    PubMed Central

    Peng, J; Harberd, N P

    1997-01-01

    Plant growth and development are regulated by numerous internal and external factors. Among these, gibberellin (GA) (an endogenous plant growth regulator) and phytochrome (a photoreceptor) often influence the same processes. For example, in plants grown in the light Arabidopsis thaliana hypocotyl elongation is reduced by GA deficiency and increased by phytochrome deficiency. Here we describe experiments in which the phenotypes of Arabidopsis plants doubly homozygous for GA-related and phytochrome-related mutations were examined. The double mutants were studied at various stages in the plant life cycle, including the seed germination, young seedling, adult, and reproductive phases of development. The results of these experiments are complex, but indicate that a fully functional GA system is necessary for full expression of the elongated phenotypes conferred by phytochrome deficiency. PMID:9112768

  18. The fine structure of elongate gametocytes of Leucocytozoon ziemanni (Laveran).

    PubMed

    Kocan, A A; Kocan, K M

    1978-12-01

    In an effort to establish comparative data within the genus Leucocytozoon, elongate gametocytes of L. ziemanni from naturally infected great horned owls (Bubo virginianus) were examined by electron microscopy. Micro- and macrogametocytes proved to be easily distinguishable at the electron microscopic level due to dramatic dimorphism at maturity and cytoplasmic and nuclear morphology. The parasite membrane architecture, number and type of cytoplasmic ribosomes of both micro- and macrogametocytes, presence and arrangement of osmiophilic bodies and electron dense spheres, mitochondrial morphology, endoplasmic reticulum cisternae morphology, mitochondria containing pocket infoldings of the nuclear membrane of the microgametocytes, and cytostome and food vacuole formation compare favorably with available information on L. simondi and L. smithi. Comparative variations exist only in that L. ziemanni gametocytes apparently lack compartmentalization of the cytoplasm by aligned unit membranes and parasite induced separations of the host cell nucleus as reported for L. simondi. PMID:105117

  19. Reconstruction of recurrent diaphragmatic eventration with an elongated polytetrafluoroethylene sheet

    PubMed Central

    Ikeda, Masaki; Sonobe, Makoto; Bando, Toru; Date, Hiroshi

    2013-01-01

    We report the case of a 31-year old woman with recurrence of left diaphragmatic eventration 3 years after a previous surgery for this condition. At the initial occurrence, she had experienced dyspnoea on exercise and subsequently underwent laparoscopic plication of the diaphragm with an endo-stapler at a local hospital. Immediately after the operation, the diaphragm was torn and the intestine entered the thorax. Therefore, plication involving sewing was performed. Then, 3 years later, the patient again experienced dyspnoea and was diagnosed as having recurrence of left diaphragmatic eventration. Observation under thoracoscopy revealed that the centre of the left diaphragm was thin but not torn. We reconstructed the left diaphragm with an elongated polytetrafluoroethylene sheet on the naïve diaphragm. The patient was discharged from our hospital 5 days after surgery. Her respiratory function improved and she has not experienced recurrence. PMID:23644727

  20. Longitudinal domain wall formation in elongated assemblies of ferromagnetic nanoparticles

    PubMed Central

    Varón, Miriam; Beleggia, Marco; Jordanovic, Jelena; Schiøtz, Jakob; Kasama, Takeshi; Puntes, Victor F.; Frandsen, Cathrine

    2015-01-01

    Through evaporation of dense colloids of ferromagnetic ~13 nm ε-Co particles onto carbon substrates, anisotropic magnetic dipolar interactions can support formation of elongated particle structures with aggregate thicknesses of 100–400 nm and lengths of up to some hundred microns. Lorenz microscopy and electron holography reveal collective magnetic ordering in these structures. However, in contrast to continuous ferromagnetic thin films of comparable dimensions, domain walls appear preferentially as longitudinal, i.e., oriented parallel to the long axis of the nanoparticle assemblies. We explain this unusual domain structure as the result of dipolar interactions and shape anisotropy, in the absence of inter-particle exchange coupling. PMID:26416297

  1. Centrosome splitting during nuclear elongation in the Drosophila embryo.

    PubMed

    Callaini, G; Anselmi, F

    1988-10-01

    In the early Drosophila embryo, nuclear elongation occurs during cellularization of the syncytial blastoderm. This process is closely related to the presence of microtubular bundles forming a basket-like structure surrounding the nuclei. In immunofluorescence observations with antibodies against alpha-tubulin, the microtubules appear to radiate from two bright foci widely separated from each other. We used electron microscopy to show that these foci are true centrosomes constituted by daughter and parent centrioles orthogonally disposed and surrounded by pericentriolar electrondense material. The centrosomes may be observed in the apical region of the blastoderm cells from the beginning of cellularization until the reestablishment of the first postblastodermic mitosis, when they organize the spindle poles. Until this time the dimensions of the procentrioles remain unchanged. The significance of these results is discussed in relation to the known behavior of centrioles in the cell cycle.

  2. Content-Based Retrieval of Medical Images with Elongated Structures

    NASA Astrophysics Data System (ADS)

    Machado, Alexei Manso Correa; Teixeira, Christiano Augusto Caldas

    In this paper we propose a set of methods to describe, register and retrieve images of elongated structures from a database based on their shape content. Registration is performed based on an elastic matching algorithm that jointly takes into account the gross shape of the structure and the shape of its boundary, resulting in anatomically consistent deformations. The method determines a medial axis that represents the full extent of the structure with no branches. Discriminative anatomic features are computed from the results of registration and used as variables in a content-based image retrieval system. A case study on the morphology of the corpus callosum in the chromosome 22q11.2 deletion syndrome illustrates the effectiveness of the method and corroborates the hypothesis that retrieval systems may also act as knowledge discovery tools.

  3. Ultraviolet spectroscopy of the zodiacal light at 20-deg elongation

    NASA Technical Reports Server (NTRS)

    Feldman, P. D.

    1977-01-01

    The zodiacal light at 20-deg elongation and 10-deg inclination was observed by rocket ultraviolet spectrometers at 10-15-A resolution in the spectral range 1200-3200 A during an experiment designed to observe comet Kohoutek (1973 XII). The data were obtained above 180 km when scattered horizon light in the startracker caused a loss of tracking on the comet. Airglow emission due to NO and O(+), identified spectroscopically and by its variation with altitude, is significant between 1900 and 2500 A. Longward of 2600 A, the spectrum matches that of the sun, and the derived value of the color ratio, relative to the visible, is 0.90 + or - 0.20. At 1600 A, an upper limit on the zodiacal-light emission of 0.07 R per A or 7 hundred-millionths erg/s per sq cm/sterad per A is obtained.

  4. Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species

    PubMed Central

    Ma, Xiqing; Huang, Bingru

    2016-01-01

    Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; ‘BR’) and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; ‘Baron’) were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

  5. Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species.

    PubMed

    Ma, Xiqing; Huang, Bingru

    2016-01-01

    Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; 'BR') and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; 'Baron') were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

  6. Pdx-1 links histone H3-Lys-4 methylation to RNA polymerase II elongation during activation of insulin transcription.

    PubMed

    Francis, Joshua; Chakrabarti, Swarup K; Garmey, James C; Mirmira, Raghavendra G

    2005-10-28

    Expression of the insulin gene is nearly exclusive to the beta cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in betaTC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.

  7. Elongational rheology and cohesive fracture of photo-oxidated LDPE

    SciTech Connect

    Rolón-Garrido, Víctor H. Wagner, Manfred H.

    2014-01-15

    It was found recently that low-density polyethylene (LDPE) samples with different degrees of photo-oxidation represent an interesting system to study the transition from ductile to cohesive fracture and the aspects of the cohesive rupture in elongational flow. Sheets of LDPE were subjected to photo-oxidation in the presence of air using a xenon lamp to irradiate the samples for times between 1 day and 6 weeks. Characterisation methods included Fourier transform infrared spectroscopy, solvent extraction method, and rheology in shear and uniaxial extensional flows. Linear viscoelasticity was increasingly affected by increasing photo-oxidation due to crosslinking of LDPE, as corroborated by the carbonyl index, acid and aldehydes groups, and gel fraction. The molecular stress function model was used to quantify the experimental data, and the nonlinear model parameter β was found to be correlated with the gel content. The uniaxial data showed that the transition from ductile to cohesive fracture was shifted to lower elongational rates, the higher the gel content was. From 2 weeks photo-oxidation onwards, cohesive rupture occurred at every strain rate investigated. The true strain and true stress at cohesive fracture as well as the energy density applied to the sample up to fracture were analyzed. At low gel content, rupture was mainly determined by the melt fraction while at high gel content, rupture occurred predominantly in the gel structure. The strain at break was found to be independent of strain rate, contrary to the stress at break and the energy density. Thus, the true strain and not the stress at break or the energy density was found to be the relevant physical quantity to describe cohesive fracture behavior of photo-oxidated LDPE. The equilibrium modulus of the gel structures was correlated with the true strain at rupture. The stiffer the gel structure, the lower was the deformation tolerated before the sample breaks.

  8. Effect of elongational flow on ferrofuids under a magnetic field.

    PubMed

    Altmeyer, S; Do, Younghae; Lopez, J M

    2013-07-01

    To set up a mathematical model for the flow of complex magnetic fluids, noninteracting magnetic particles with a small volume or an even point size are typically assumed. Real ferrofluids, however, consist of a suspension of particles with a finite size in an almost ellipsoid shape as well as with particle-particle interactions that tend to form chains of various lengths. To come close to the realistic situation for ferrofluids, we investigate the effect of elongational flow incorporated by the symmetric part of the velocity gradient field tensor, which could be scaled by a so-called transport coefficient λ(2). Based on the hybrid finite-difference and Galerkin scheme, we study the flow of a ferrofluid in the gap between two concentric rotating cylinders subjected to either a transverse or an axial magnetic field with the transport coefficient. Under the influence of a transverse magnetic field with λ(2)=0, we show that basic state and centrifugal unstable flows are modified and are inherently three-dimensional helical flows that are either left-winding or right-winding in the sense of the azimuthal mode-2, which is in contrast to the generic cases. That is, classical modulated rotating waves rotate, but these flows do not. We find that under elongational flow (λ(2)≠0), the flow structure from basic state and centrifugal instability flows is modified and their azimuthal vorticity is linearly changed. In addition, we also show that the bifurcation threshold of the supercritical centrifugal unstable flows under a magnetic field depends linearly on the transport coefficient, but it does not affect the general stabilization effect of any magnetic field. PMID:23944545

  9. Three-dimensional structure of photosystem II from Thermosynechococcus elongates in complex with terbutryn

    SciTech Connect

    Gabdulkhakov, A. G. Dontsova, M. V.; Saenger, W.

    2011-11-15

    Photosystem II is a key component of the photosynthetic pathway producing oxygen at the thylakoid membrane of cyanobacteria, green algae, and plants. The three-dimensional structure of photosystem II from the cyanobacterium Thermosynechococcus elongates in a complex with herbicide terbutryn (a photosynthesis inhibitor) was determined for the first time by X-ray diffraction and refined at 3.2 Angstrom-Sign resolution (R{sub factor} = 26.9%, R{sub free} = 29.9%, rmsd for bond lengths is 0.013 Angstrom-Sign , and rmsd for bond angles is 2.2 Degree-Sign ). The terbutryn molecule was located in the binding pocket of the mobile plastoquinone. The atomic coordinates of the refined structure of photosystem II in a complex with terbutryn were deposited in the Protein Data Bank.

  10. Coping with asymmetry: How infants and adults walk with one elongated leg

    PubMed Central

    Cole, Whitney G.; Gill, Simone V.; Vereijken, Beatrix; Adolph, Karen E.

    2014-01-01

    The stability of a system affects how it will handle a perturbation: The system may compensate for the perturbation or not. This study examined how 14-month-old infants—notoriously unstable walkers—and adults cope with a perturbation to walking. We attached a platform to one of participants' shoes, forcing them to walk with one elongated leg. At first, the platform shoe caused both age groups to slow down and limp, and caused infants to misstep and fall. But after a few trials, infants altered their gait to compensate for the platform shoe whereas adults did not; infants recovered symmetrical gait whereas adults continued to limp. Apparently, adult walking was stable enough to cope with the perturbation, but infants risked falling if they did not compensate. Compensation depends on the interplay of multiple factors: The availability of a compensatory response, the cost of compensation, and the stability of the system being perturbed. PMID:24857934

  11. Effect of the Acceleration of Elongated Bodies of Revolution Upon the Resistance in a Compressible Flow

    NASA Technical Reports Server (NTRS)

    Frankl, F. I.

    1949-01-01

    The problem of the motion of an elongated body of revolution in an incompressible fluid may, as is known, be solved approximately with the aid of the distribution of sources along the axis of the body. In determining the velocity field, the question of whether the body moves uniformly or with an acceleration is no factor in the problem. The presence of acceleration must be taken into account in determining the pressures acting on the body. The resistance of the body arising from the accelerated motion may be computed either directly on the basis of these pressures or with the aid of the so-called associated masses (inertia coefficients). A different condition holds in the case of the motion of bodies in a compressible gas. In this case the finite velocity of sound must be taken into account.

  12. Defective Dendrite Elongation but Normal Fertility in Mice Lacking the Rho-Like GTPase Activator Dbl

    PubMed Central

    Hirsch, Emilio; Pozzato, Michela; Vercelli, Alessandro; Barberis, Laura; Azzolino, Ornella; Russo, Chiara; Vanni, Cristina; Silengo, Lorenzo; Eva, Alessandra; Altruda, Fiorella

    2002-01-01

    Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation. PMID:11940671

  13. Drosophila Strip serves as a platform for early endosome organization during axon elongation

    PubMed Central

    Sakuma, Chisako; Kawauchi, Takeshi; Haraguchi, Shuka; Shikanai, Mima; Yamaguchi, Yoshifumi; Gelfand, Vladimir I.; Luo, Liqun; Miura, Masayuki; Chihara, Takahiro

    2014-01-01

    Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. Here we show that Drosophila Strip is a key regulator for neuronal morphogenesis. strip knockdown disturbs the early endosome clustering and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that plays important roles in neuronal morphogenesis. PMID:25312435

  14. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    PubMed

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-01

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

  15. The Acid Growth Theory of auxin-induced cell elongation is alive and well

    NASA Technical Reports Server (NTRS)

    Rayle, D. L.; Cleland, R. E.

    1992-01-01

    Plant cells elongate irreversibly only when load-bearing bonds in the walls are cleaved. Auxin causes the elongation of stem and coleoptile cells by promoting wall loosening via cleavage of these bonds. This process may be coupled with the intercalation of new cell wall polymers. Because the primary site of auxin action appears to be the plasma membrane or some intracellular site, and wall loosening is extracellular, there must be communication between the protoplast and the wall. Some "wall-loosening factor" must be exported from auxin-impacted cells, which sets into motion the wall loosening events. About 20 years ago, it was suggested that the wall-loosening factor is hydrogen ions. This idea and subsequent supporting data gave rise to the Acid Growth Theory, which states that when exposed to auxin, susceptible cells excrete protons into the wall (apoplast) at an enhanced rate, resulting in a decrease in apoplastic pH. The lowered wall pH then activates wall-loosening processes, the precise nature of which is unknown. Because exogenous acid causes a transient (1-4 h) increase in growth rate, auxin must also mediate events in addition to wall acidification for growth to continue for an extended period of time. These events may include osmoregulation, cell wall synthesis, and maintenance of the capacity of walls to undergo acid-induced wall loosening. At present, we do not know if these phenomena are tightly coupled to wall acidification or if they are the products of multiple independent signal transduction pathways.

  16. Phytotoxicity of veterinary antibiotics to seed germination and root elongation of crops.

    PubMed

    Pan, Min; Chu, L M

    2016-04-01

    Large quantities of veterinary antibiotics (VAs) are being used worldwide in agricultural fields through wastewater irrigation and manure application. They cause damages to the ecosystem when discharged into the environment, but there is a lack of information on their toxicity to plants and animals. This study evaluated the phytotoxic effects of five major VAs, namely tetracycline (TC), sulfamethazine (SMZ), norfloxacin (NOR), erythromycin (ERY) and chloramphenicol (CAP), on seed germination and root elongation in lettuce, tomato, carrot and cucumber, and investigated the relationship between their physicochemical properties and phytotoxicities. Results show that these compounds significantly inhibited root elongation (p<0.05), the most sensitive endpoint for the phytotoxicity test. TC was associated with the highest level of toxicity, followed by NOR, ERY, SMZ and CAP. Regarding crop species, lettuce was found to be sensitive to most of the VAs. The median effect concentration (EC50) of TC, SMZ, NOR, ERY and CAP to lettuce was 14.4, 157, 49.4, 68.8 and 204 mg/L, respectively. A quantitative structure-activity relationship (QSAR) model has been established based on the measured data. It is evident that hydrophobicity was the most important factor governing the phytotoxicity of these compounds to seeds, which could be explained by the polar narcosis mechanism. Lettuce is considered a good biomarker for VAs in the environment. According to the derived equation, phytotoxicities of selected VA compounds on different crops can be calculated, which could be applicable to other VAs. Environmental risks of VAs were summarized based on the phytotoxicity results and other persistent factors. PMID:26773832

  17. Phytotoxicity of veterinary antibiotics to seed germination and root elongation of crops.

    PubMed

    Pan, Min; Chu, L M

    2016-04-01

    Large quantities of veterinary antibiotics (VAs) are being used worldwide in agricultural fields through wastewater irrigation and manure application. They cause damages to the ecosystem when discharged into the environment, but there is a lack of information on their toxicity to plants and animals. This study evaluated the phytotoxic effects of five major VAs, namely tetracycline (TC), sulfamethazine (SMZ), norfloxacin (NOR), erythromycin (ERY) and chloramphenicol (CAP), on seed germination and root elongation in lettuce, tomato, carrot and cucumber, and investigated the relationship between their physicochemical properties and phytotoxicities. Results show that these compounds significantly inhibited root elongation (p<0.05), the most sensitive endpoint for the phytotoxicity test. TC was associated with the highest level of toxicity, followed by NOR, ERY, SMZ and CAP. Regarding crop species, lettuce was found to be sensitive to most of the VAs. The median effect concentration (EC50) of TC, SMZ, NOR, ERY and CAP to lettuce was 14.4, 157, 49.4, 68.8 and 204 mg/L, respectively. A quantitative structure-activity relationship (QSAR) model has been established based on the measured data. It is evident that hydrophobicity was the most important factor governing the phytotoxicity of these compounds to seeds, which could be explained by the polar narcosis mechanism. Lettuce is considered a good biomarker for VAs in the environment. According to the derived equation, phytotoxicities of selected VA compounds on different crops can be calculated, which could be applicable to other VAs. Environmental risks of VAs were summarized based on the phytotoxicity results and other persistent factors.

  18. Anisotropic Resist Reflow Process Simulation for 22 nm Elongated Contact Holes

    NASA Astrophysics Data System (ADS)

    Park, Joon-Min; Kim, Dai-Gyoung; Hong, Joo-Yoo; An, Ilsin; Oh, Hye-Keun

    2008-06-01

    Pattern size decreases as circuit integration increases. Resistance increases as the cross section of a contact hole (CH) decreases. Thus, the use of an elongated CH is suggested as a method of solving this problem. It is too difficult to obtain a small CH and an elongated CH by optical proximity correction only. Even if double patterning can be used to improve the integration of line and space, it is not easy to apply it to form an elongated CH. We suggest the use of a resist reflow process method to form 22 nm elongated CHs from a large developed size pattern. We observed RRP behavior in elongated CHs by experiment and simulation, and applied optical proximity correction to compensate the bulk effect after the resist reflow process. As a result, we made uniform 22 nm elongated CHs.

  19. Genetic separation of phototropism from blue light inhibition of hypocotyl elongation on Arabidopsis

    SciTech Connect

    Liscum, E.; Young, J.C.; Hangarter, R.P. ); Poff, K.L. )

    1991-05-01

    Phototropism and inhibition of stem elongation occur in response to blue light-induced inhibition of cell elongation. However, phototropism is a low fluence response and inhibition of hypocotyl elongation is a high irradiance response. The authors have isolated several mutant lines of Arabidopsis which lack blue light-induced inhibition of hypocotyl elongation but retain normal phototropic functions. In addition, a mutant line which completely lacks the phototropic response retains normal blue light-induced inhibition of hypocotyl elongation. F1 progeny of crosses between these two mutant classes exhibited wild-type phototropism and inhibition of hypocotyl elongation in response to blue light stimuli. In the F2 generation, one in sixteen seedlings were double mutants lacking both phototropism and blue light-induced hypocotyl growth inhibition. These studies conclusively show that blue light-induced phototropism and hypocotyl growth inhibition function through genetically distinct signal transduction or response systems.

  20. Elongation Kinetics of Polyglutamine Peptide Fibrils: A Quartz Crystal Microbalance with Dissipation Study

    PubMed Central

    Walters, Robert H.; Jacobson, Kurt H.; Pedersen, Joel A.; Murphy, Regina M.

    2012-01-01

    Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation. PMID:22459263

  1. Elongated styloid process in a temporomandibular disorder sample: prevalence and treatment outcome.

    PubMed

    Zaki, H S; Greco, C M; Rudy, T E; Kubinski, J A

    1996-04-01

    An elongated styloid process is an anatomic anomaly present in 2% to 30% of adults; it is occasionally associated with pain. Its prevalence among patients with classic temporomandibular disorder pain symptoms is unknown. The effect of conservative treatment on patients who have symptoms of temporomandibular disorders and an elongated styloid process is also unknown. The objectives of this study were to determine the prevalence of the elongated styloid process in a sample of patients with temporomandibular disorders and to compare patients with and without the elongated styloid process on initial presenting signs and symptoms and treatment outcome. A total of 100 panoramic radiographs of patients with symptomatic temporomandibular disorders were examined to ascertain the presence or absence of an elongated styloid process. All patients participated in a conservative treatment program of biofeedback and stress management and a flat-plane intraoral appliance. Initial symptoms and treatment outcome of patients with and without an elongated styloid process were compared by use of multivariate analysis of variance on several oral-paraoral and psychosocial-behavioral methods. The prevalence of an elongated styloid process in this clinic sample of temporomandibular disorders was 27%. The patients with or without an elongated styloid process were not significantly different in pretreatment symptoms, and both groups exhibited substantial treatment gains. However, patients with an elongated styloid process showed significantly less improvement on unassisted mandibular opening without pain than did patients who did not have an elongated styloid process. This suggests that an elongated styloid process may place structural limitations on pain-free maximum mandibular opening. The results support conservative management of patients with symptoms of temporomandibular disorders when an elongated styloid process is present.

  2. Structure of the FMNL3 FH2/actin complex provides insight into formin-mediated actin nucleation and elongation

    PubMed Central

    Thompson, Morgan E.; Heimsath, Ernest G.; Gauvin, Timothy J.; Higgs, Henry N.; Kull, F. Jon

    2012-01-01

    Summary Formins are actin assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation via interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4 Å structure of an FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2-actin binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding while those in the lasso/post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2 mediated filament elongation via processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus. PMID:23222643

  3. The IKAROS Interaction with a Complex Including Chromatin Remodeling and Transcription Elongation Activities Is Required for Hematopoiesis

    PubMed Central

    Bottardi, Stefania; Mavoungou, Lionel; Pak, Helen; Daou, Salima; Bourgoin, Vincent; Lakehal, Yahia A.; Affar, El Bachir; Milot, Eric

    2014-01-01

    IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation. PMID:25474253

  4. Zonisamide Enhances Neurite Elongation of Primary Motor Neurons and Facilitates Peripheral Nerve Regeneration In Vitro and in a Mouse Model

    PubMed Central

    Yagi, Hideki; Ohkawara, Bisei; Nakashima, Hiroaki; Ito, Kenyu; Tsushima, Mikito; Ishii, Hisao; Noto, Kimitoshi; Ohta, Kyotaro; Masuda, Akio; Imagama, Shiro; Ishiguro, Naoki; Ohno, Kinji

    2015-01-01

    No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies. PMID:26571146

  5. Integrated metabolomics and genomics analysis provides new insights into the fiber elongation process in Ligon lintless-2 mutant cotton (Gossypium hirsutum L.)

    PubMed Central

    2013-01-01

    Background The length of cotton fiber is an important agronomic trait characteristic that directly affects the quality of yarn and fabric. The cotton (Gossypium hirsutum L.) fiber mutation, Ligon lintless-2, is controlled by a single dominant gene (Li2) and results in extremely shortened lint fibers on mature seeds with no visible pleiotropic effects on vegetative growth and development. The Li2 mutant phenotype provides an ideal model system to study fiber elongation. To understand metabolic processes involved in cotton fiber elongation, changes in metabolites and transcripts in the Li2 mutant fibers were compared to wild-type fibers during development. Results Principal component analysis of metabolites from GC-MS data separated Li2 mutant fiber samples from WT fiber samples at the WT elongation stage, indicating that the Li2 mutation altered the metabolome of the mutant fibers. The observed alterations in the Li2 metabolome included significant reductions in the levels of detected free sugars, sugar alcohols, sugar acids, and sugar phosphates. Biological processes associated with carbohydrate biosynthesis, cell wall loosening, and cytoskeleton were also down-regulated in Li2 fibers. Gamma-aminobutyric acid, known as a signaling factor in many organisms, was significantly elevated in mutant fibers. Higher accumulation of 2-ketoglutarate, succinate, and malate suggested higher nitrate assimilation in the Li2 line. Transcriptional activation of genes involved in nitrogen compound metabolism along with changes in the levels of nitrogen transport amino acids suggested re-direction of carbon flow into nitrogen metabolism in Li2 mutant fibers. Conclusions This report provides the first comprehensive analysis of metabolite and transcript changes in response to the Li2 mutation in elongating fibers. A number of factors associated with cell elongation found in this study will facilitate further research in understanding metabolic processes of cotton fiber elongation. PMID

  6. Shear banding in titanium with controlled elongations at 10/sup 6//sec

    SciTech Connect

    Staudhammer, K.P.; Gray, A.J.

    1989-01-01

    Adiabatic shear bands were obtained in titanium with controlled strains to 10.8%. Two types of shear bands were observed and characterized. In general the quantity and shear band width increased with increasing elongation. At elongations below /approximately/5% the shear bands were typically characteristic of shear bands observed at lower strain rates. Above 5% elongations many of the shear bands contained a boundary regime adjacent to the shear band and increased in width with increasing total elongation. This effect was attributed to the strain heat. 14 refs., 9 figs.

  7. Actin filaments and microtubules play different roles during bristle elongation in Drosophila.

    PubMed

    Tilney, L G; Connelly, P S; Vranich, K A; Shaw, M K; Guild, G M

    2000-04-01

    Developing bristles in Drosophila pupae contain 7-11 bundles of crosslinked actin filaments and a large population of microtubules. During bristle growth the rate of cell elongation increases with bristle length. Thin section EM shows that bundle size is correlated with the amount of cytoplasm at all points along the bristle. Thus, as the bristle elongates and tapers, fewer actin filaments are used. To ensure penetration of inhibitors we isolated thoraces and cultured them in vitro; bristles elongate at rates identical to bristles growing in situ. Interestingly, inhibitors of actin filament assembly (cytochalasin D and latrunculin A) dramatically curtailed bristle elongation while a filament stabilizer (jasplakinolide) accelerated elongation. In contrast, inhibitors of microtubule dynamics (nocodazole, vinblastine, colchicine and taxol) did not affect bristle elongation. Surprisingly, the bristle microtubules are stable and do not turn over. Furthermore, the density of microtubules decreases as the bristle elongates. These two facts coupled with calculations and kinetics of elongation and the fact that the microtubules are short indicate that the microtubules are assembled early in development and then transported distally as the bristle grows. We conclude that actin assembly is crucial for bristle cell elongation and that microtubules must furnish other functions such as to provide bulk to the bristle cytoplasm as well as playing a role in vesicle transport.

  8. Fitness consequences of natural variation in flooding-induced shoot elongation in Rumex palustris.

    PubMed

    Chen, Xin; Visser, Eric J W; de Kroon, Hans; Pierik, Ronald; Voesenek, Laurentius A C J; Huber, Heidrun

    2011-04-01

    • Plants can respond to their environment by morphological plasticity. Generally, the potential benefits of adaptive plastic responses are beyond doubt under predictable environmental changes. However, the net benefits may be less straightforward when plants encounter temporal stresses, such as flooding in river flood plains. • Here, we tested whether the balance of costs and benefits associated with flooding-induced shoot elongation depends on the flooding regime, by subjecting Rumex palustris plants with different elongation capacity to submergence of different frequency and duration. • Our results showed that reaching the surface by shoot elongation is associated with fitness benefits, as under less frequent, but longer, flooding episodes plants emerging above the floodwater had greater biomass production than plants that were kept below the surface. As we predicted, slow-elongating plants had clear advantages over fast-elongating ones if submergence was frequent but of short duration, indicating that elongation also incurs costs. • Our data suggest that high costs select for weak plasticity under frequent environmental change. In contrast to our predictions, however, fast-elongating plants did not have an overall advantage over slow-elongating plants when floods lasted longer. This indicates that the delicate balance between benefits and costs of flooding-induced elongation depends on the specific characteristics of the flooding regime.

  9. Micro- and Nanoscale Capacitors that Incorporate an Array of Conductive Elements Having Elongated Bodies

    NASA Technical Reports Server (NTRS)

    Manohara, Harish (Inventor); Del Castillo, Linda Y. (Inventor); Mojarradi, Mohammed M. (Inventor)

    2016-01-01

    Systems and methods in accordance with embodiments of the invention implement micro- and nanoscale capacitors that incorporate a conductive element that conforms to the shape of an array elongated bodies. In one embodiment, a capacitor that incorporates a conductive element that conforms to the shape of an array of elongated bodies includes: a first conductive element that conforms to the shape of an array of elongated bodies; a second conductive element that conforms to the shape of an array of elongated bodies; and a dielectric material disposed in between the first conductive element and the second conductive element, and thereby physically separates them.

  10. Application of an Elongated Kelvin Model to Space Shuttle Foams

    NASA Technical Reports Server (NTRS)

    Sullivan, Roy M.; Ghosn, Louis J.; Lerch, Bradley A.

    2008-01-01

    Spray-on foam insulation is applied to the exterior of the Space Shuttle s External Tank to limit propellant boil-off and to prevent ice formation. The Space Shuttle foams are rigid closed-cell polyurethane foams. The two foams used most extensively on the Space Shuttle External Tank are BX-265 and NCFI24-124. Since the catastrophic loss of the Space Shuttle Columbia, numerous studies have been conducted to mitigate the likelihood and the severity of foam shedding during the Shuttle s ascent to space. Due to the foaming and rising process, the foam microstructures are elongated in the rise direction. As a result, these two foams exhibit a non-isotropic mechanical behavior. In this paper, a detailed microstructural characterization of the two foams is presented. The key features of the foam cells are summarized and the average cell dimensions in the two foams are compared. Experimental studies to measure the room temperature mechanical response of the two foams in the two principal material directions (parallel to the rise and perpendicular to the rise) are also reported. The measured elastic modulus, proportional limit stress, ultimate tensile stress and the Poisson s ratios for the two foams are compared. The generalized elongated Kelvin foam model previously developed by the authors is reviewed and the equations which result from this model are presented. The resulting equations show that the ratio of the elastic modulus in the rise direction to that in the perpendicular-to-rise direction as well as the ratio of the strengths in the two material directions is only a function of the microstructural dimensions. Using the measured microstructural dimensions and the measured stiffness ratio, the foam tensile strength ratio and Poisson s ratios are predicted for both foams. The predicted tensile strength ratio is in close agreement with the measured strength ratios for both BX-265 and NCFI24-124. The comparison between the predicted Poisson s ratios and the measured

  11. Gibberellin A[sub 1] is required for stem elongation in spinach

    SciTech Connect

    Zeevaart, J.A.D.; Gage, D.A.; Talon, M. )

    1993-08-01

    The effects of the growth retardants 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (AMO-1618) and calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate (BX-112) on stem elongation were investigated in the rosette plant spinach (Spinacia oleracea L.) under long-day (LD) conditions. Stem growth induced by a LD treatment was prevented by both retardants. The inhibition caused by AMO-1618 was reversed by gibberellin A[sub 1] (GA[sub 1]) and GA[sub 20], whereas the effects of BX-112 were reversed by GA[sub 1] only. Six GAs (GA[sub 53], GA[sub 44], GA[sub 19], GA[sub 20], GA[sub 1], and GA[sub 8]) were quantified by gas chromatography-selected ion monitoring using internal standards. Plants treated with BX-112 had reduced levels of GA[sub 1], and GA[sub 8] and accumulated GA[sub 53], GA[sub 44], GA[sub 19], and GA[sub 20]. The relative levels of four additional GAs (3-epi-GA[sub 1], GA[sub 29], GA[sub 60], and GA[sub 81]) were compared by ion intensities only. Relative to GA[sub 81], the level of GA[sub 29] was decreased by BX-112, whereas the levels of GA[sub 6] and 3-epi-GA[sub 1] were increased. Transfer of spinach from short-day conditions to LD conditions caused an increase in all identified GAs of the early 13-hydroxylation pathway with GA[sub 20], GA[sub 1], and GA[sub 8] showing the largest increases. These findings support the position that, of the GA[sub s] belonging to the early 13-hydroxylation pathway, GA[sub 1] is the primary GA active per se for stem elongation in spinach. The increase in endogenous GA[sub 1] in plants in LD conditions is most likely the primary factor for stem elongation. 23 refs., 7 figs., 3 tabs.

  12. Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.

    PubMed

    Ribeiro, Filipa F; Neves-Tomé, Raquel; Assaife-Lopes, Natália; Santos, Telma E; Silva, Rui F M; Brites, Dora; Ribeiro, Joaquim A; Sousa, Mónica M; Sebastião, Ana M

    2016-06-01

    Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration.

  13. Phosphorus and magnesium interactively modulate the elongation and directional growth of primary roots in Arabidopsis thaliana (L.) Heynh

    PubMed Central

    Niu, Yaofang; Jin, Gulei; Li, Xin; Tang, Caixian; Zhang, Yongsong; Liang, Yongchao; Yu, Jingquan

    2015-01-01

    A balanced supply of essential nutrients is an important factor influencing root architecture in many plants, yet data related to the interactive effects of two nutrients on root growth are limited. Here, we investigated the interactive effect between phosphorus (P) and magnesium (Mg) on root growth of Arabidopsis grown in pH-buffered agar medium at different P and Mg levels. The results showed that elongation and deviation of primary roots were directly correlated with the amount of P added to the medium but could be modified by the Mg level, which was related to the root meristem activity and stem-cell division. High P enhanced while low P decreased the tip-focused fluorescence signal of auxin biosynthesis, transport, and redistribution during elongation of primary roots; these effects were greater under low Mg than under high Mg. The altered root growth in response to P and Mg supply was correlated with AUX1, PIN2, and PIN3 mRNA abundance and expression and the accumulation of the protein. Application of either auxin influx inhibitor or efflux inhibitor inhibited the elongation and increased the deviation angle of primary roots, and decreased auxin level in root tips. Furthermore, the auxin-transport mutants aux1-22 and eir1-1 displayed reduced root growth and increased the deviation angle. Our data suggest a profound effect of the combined supply of P and Mg on the development of root morphology in Arabidopsis through auxin signals that modulate the elongation and directional growth of primary root and the expression of root differentiation and development genes. PMID:25922494

  14. Phosphorus and magnesium interactively modulate the elongation and directional growth of primary roots in Arabidopsis thaliana (L.) Heynh.

    PubMed

    Niu, Yaofang; Jin, Gulei; Li, Xin; Tang, Caixian; Zhang, Yongsong; Liang, Yongchao; Yu, Jingquan

    2015-07-01

    A balanced supply of essential nutrients is an important factor influencing root architecture in many plants, yet data related to the interactive effects of two nutrients on root growth are limited. Here, we investigated the interactive effect between phosphorus (P) and magnesium (Mg) on root growth of Arabidopsis grown in pH-buffered agar medium at different P and Mg levels. The results showed that elongation and deviation of primary roots were directly correlated with the amount of P added to the medium but could be modified by the Mg level, which was related to the root meristem activity and stem-cell division. High P enhanced while low P decreased the tip-focused fluorescence signal of auxin biosynthesis, transport, and redistribution during elongation of primary roots; these effects were greater under low Mg than under high Mg. The altered root growth in response to P and Mg supply was correlated with AUX1, PIN2, and PIN3 mRNA abundance and expression and the accumulation of the protein. Application of either auxin influx inhibitor or efflux inhibitor inhibited the elongation and increased the deviation angle of primary roots, and decreased auxin level in root tips. Furthermore, the auxin-transport mutants aux1-22 and eir1-1 displayed reduced root growth and increased the deviation angle. Our data suggest a profound effect of the combined supply of P and Mg on the development of root morphology in Arabidopsis through auxin signals that modulate the elongation and directional growth of primary root and the expression of root differentiation and development genes.

  15. Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch.

    PubMed

    Harvey, Beth M; Rana, Nadia A; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S

    2016-07-29

    Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.

  16. The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation.

    PubMed

    Ghorbani, Sarieh; Hoogewijs, Kurt; Pečenková, Tamara; Fernandez, Ana; Inzé, Annelies; Eeckhout, Dominique; Kawa, Dorota; De Jaeger, Geert; Beeckman, Tom; Madder, Annemieke; Van Breusegem, Frank; Hilson, Pierre

    2016-08-01

    The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor. PMID:27315833

  17. Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch.

    PubMed

    Harvey, Beth M; Rana, Nadia A; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S

    2016-07-29

    Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

  18. The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation

    PubMed Central

    Ghorbani, Sarieh; Hoogewijs, Kurt; Pečenková, Tamara; Fernandez, Ana; Inzé, Annelies; Eeckhout, Dominique; Kawa, Dorota; De Jaeger, Geert; Beeckman, Tom; Madder, Annemieke; Van Breusegem, Frank; Hilson, Pierre

    2016-01-01

    The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo. Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor. PMID:27315833

  19. Allen's rule revisited: temperature influences bone elongation during a critical period of postnatal development.

    PubMed

    Serrat, Maria A

    2013-10-01

    Limbs of animals raised at warm ambient temperature are significantly and permanently longer than those of siblings housed in the cold. These highly reproducible lab results closely parallel the ecogeographical tenet described by Allen's extremity size rule, which states that appendage length correlates with temperature and latitude. It is unclear what mechanisms underlie these differences and in what pattern they emerge, since the morphology is traditionally thought to reflect naturally selected genomic adaptations for thermoregulation. This study tests the a posteriori hypothesis that adult extremity length is subject to substantial modification by temperature during a brief but critical period of early postnatal development. Weanling mice (N = 28) were divided into three groups and housed at 7°C, 21°C, or 27°C for eight weeks. Tail lengths and body mass were measured weekly. Mass did not differ at any age. Analysis of tail elongation curves revealed two distinct phases: an initial period of rapid temperature-sensitive growth in which elongation rate was directly impacted by temperature; and a second phase of continued growth in which rates were identical among groups. Comparable growth reactions occur in response to other environmental variables such as exercise, suggesting that the skeleton is most responsive to external stimuli during a window of heightened sensitivity when growth occurs most rapidly. Knowledge of the timing and degree to which growth plasticity permits mammals to immediately adjust to novel temperature conditions will be important for analyzing skeletal variation in fluctuating climates, particularly for assessing factors that may accelerate skeletal evolution at temperature extremes.

  20. Combining orthogonal polarization for elongated target detection with GPR

    NASA Astrophysics Data System (ADS)

    Lualdi, Maurizio; Lombardi, Federico

    2014-10-01

    For an accurate imaging of ground penetrating radar data the polarization characteristics of the propagating electromagnetic (EM) wavefield and wave amplitude variations with antenna pattern orientation must be taken into account. For objects that show some directionality feature and cylindrical shape any misalignment between transmitter and target can strongly modify the polarization state of the backscattered wavefield, thus conditioning the detection capability of the system. Hints on the depolarization can be used to design the optimal GPR antenna survey to avoid omissions and pitfalls during data processing. This research addresses the issue of elongated target detection through a multi azimuth (or multi polarization) approach based on the combination of mutually orthogonal GPR data. Results from the analysis of the formal scattering problem demonstrate how this strategy can reach a scalar formulation of the scattering matrix and achieve a rotational invariant quantity. The effectiveness of the algorithm is then evaluated with a detailed field example showing results closely proximal to those obtained under the optimal alignment condition: detection is significantly improved and the risk of target missing is reduced.