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Sample records for elongation factor 1a

  1. 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

  2. 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

  3. 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.

  4. 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...

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. Yeast translation elongation factor-1A binds vacuole-localized Rho1p to facilitate membrane integrity through F-actin remodeling.

    PubMed

    Bodman, James A R; Yang, Yang; Logan, Michael R; Eitzen, Gary

    2015-02-20

    Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. A complex distribution of elongation family GTPases EF1A and EFL in basal alveolate lineages.

    PubMed

    Mikhailov, Kirill V; Janouškovec, Jan; Tikhonenkov, Denis V; Mirzaeva, Gulnara S; Diakin, Andrei Yu; Simdyanov, Timur G; Mylnikov, Alexander P; Keeling, Patrick J; Aleoshin, Vladimir V

    2014-09-01

    Translation elongation factor-1 alpha (EF1A) and the related GTPase EF-like (EFL) are two proteins with a complex mutually exclusive distribution across the tree of eukaryotes. Recent surveys revealed that the distribution of the two GTPases in even closely related taxa is frequently at odds with their phylogenetic relationships. Here, we investigate the distribution of EF1A and EFL in the alveolate supergroup. Alveolates comprise three major lineages: ciliates and apicomplexans encode EF1A, whereas dinoflagellates encode EFL. We searched transcriptome databases for seven early-diverging alveolate taxa that do not belong to any of these groups: colpodellids, chromerids, and colponemids. Current data suggest all seven are expected to encode EF1A, but we find three genera encode EFL: Colpodella, Voromonas, and the photosynthetic Chromera. Comparing this distribution with the phylogeny of alveolates suggests that EF1A and EFL evolution in alveolates cannot be explained by a simple horizontal gene transfer event or lineage sorting.

  10. A complex distribution of elongation family GTPases EF1A and EFL in basal alveolate lineages.

    PubMed

    Mikhailov, Kirill V; Janouškovec, Jan; Tikhonenkov, Denis V; Mirzaeva, Gulnara S; Diakin, Andrei Yu; Simdyanov, Timur G; Mylnikov, Alexander P; Keeling, Patrick J; Aleoshin, Vladimir V

    2014-09-01

    Translation elongation factor-1 alpha (EF1A) and the related GTPase EF-like (EFL) are two proteins with a complex mutually exclusive distribution across the tree of eukaryotes. Recent surveys revealed that the distribution of the two GTPases in even closely related taxa is frequently at odds with their phylogenetic relationships. Here, we investigate the distribution of EF1A and EFL in the alveolate supergroup. Alveolates comprise three major lineages: ciliates and apicomplexans encode EF1A, whereas dinoflagellates encode EFL. We searched transcriptome databases for seven early-diverging alveolate taxa that do not belong to any of these groups: colpodellids, chromerids, and colponemids. Current data suggest all seven are expected to encode EF1A, but we find three genera encode EFL: Colpodella, Voromonas, and the photosynthetic Chromera. Comparing this distribution with the phylogeny of alveolates suggests that EF1A and EFL evolution in alveolates cannot be explained by a simple horizontal gene transfer event or lineage sorting. PMID:25179686

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  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. 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

  9. 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

  10. 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

  11. The Dual Functions of WLIM1a in Cell Elongation and Secondary Wall Formation in Developing Cotton Fibers[C][W

    PubMed Central

    Han, Li-Bo; Li, Yuan-Bao; Wang, Hai-Yun; Wu, Xiao-Min; Li, Chun-Li; Luo, Ming; Wu, Shen-Jie; Kong, Zhao-Sheng; Pei, Yan; Jiao, Gai-Li; Xia, Gui-Xian

    2013-01-01

    LIN-11, Isl1 and MEC-3 (LIM)-domain proteins play pivotal roles in a variety of cellular processes in animals, but plant LIM functions remain largely unexplored. Here, we demonstrate dual roles of the WLIM1a gene in fiber development in upland cotton (Gossypium hirsutum). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages in developing fibers. Overexpression of WLIM1a in cotton led to significant changes in fiber length and secondary wall structure. Compared with the wild type, fibers of WLIM1a-overexpressing plants grew longer and formed a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness. Functional studies demonstrated that (1) WLIM1a acts as an actin bundler to facilitate elongation of fiber cells and (2) WLIM1a also functions as a transcription factor to activate expression of Phe ammonia lyase–box genes involved in phenylpropanoid biosynthesis to build up the secondary cell wall. WLIM1a localizes in the cytosol and nucleus and moves into the nucleus in response to hydrogen peroxide. Taken together, these results demonstrate that WLIM1a has dual roles in cotton fiber development, elongation, and secondary wall formation. Moreover, our study shows that lignin/lignin-like phenolics may substantially affect cotton fiber quality; this finding may guide cotton breeding for improved fiber traits. PMID:24220634

  12. Brevis plant1, a putative inositol polyphosphate 5-phosphatase, is required for internode elongation in maize

    PubMed Central

    Avila, Luis M.; Cerrudo, Diego; Swanton, Clarence

    2016-01-01

    In maize (Zea mays L.), as in other grass species, stem elongation occurs during growth and most noticeably upon the transition to flowering. Genes that reduce stem elongation have been important to reduce stem breakage, or lodging. Stem elongation has been mediated by dwarf and brachytic/brevis plant mutants that affect giberellic acid and auxin pathways, respectively. Maize brevis plant1 (bv1) mutants, first identified over 80 years ago, strongly resemble brachytic2 mutants that have shortened internodes, short internode cells, and are deficient in auxin transport. Here, we characterized two novel bv1 maize mutants. We found that an inositol polyphosphate 5-phosphatase orthologue of the rice gene dwarf50 was the molecular basis for the bv1 phenotype, implicating auxin-mediated inositol polyphosphate and/or phosphoinositide signalling in stem elongation. We suggest that auxin-mediated internode elongation involves processes that also contribute to stem gravitropism. Genes misregulated in bv1 mutants included genes important for cell wall synthesis, transmembrane transport, and cytoskeletal function. Mutant and wild-type plants were indistinguishable early in development, responded similarly to changes in light quality, had unaltered flowering times, and had normal flower development. These attributes suggest that breeding could utilize bv1 alleles to increase crop grain yields. PMID:26767748

  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 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

  15. 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.

  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. 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

  19. 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.

  20. 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 ...

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation.

    PubMed

    Lv, Fenni; Wang, Haihai; Wang, Xinyu; Han, Libo; Ma, Yinping; Wang, Sen; Feng, Zhidi; Niu, Xiaowei; Cai, Caiping; Kong, Zhaosheng; Zhang, Tianzhen; Guo, Wangzhen

    2015-04-01

    Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Elongated Structure of the Outer-Membrane Activator of Peptidoglycan Synthesis LpoA: Implications for PBP1A Stimulation

    PubMed Central

    Jean, Nicolas L.; Bougault, Catherine M.; Lodge, Adam; Derouaux, Adeline; Callens, Gilles; Egan, Alexander J.F.; Ayala, Isabel; Lewis, Richard J.; Vollmer, Waldemar; Simorre, Jean-Pierre

    2014-01-01

    Summary The bacterial cell envelope contains the stress-bearing peptidoglycan layer, which is enlarged during cell growth and division by membrane-anchored synthases guided by cytoskeletal elements. In Escherichia coli, the major peptidoglycan synthase PBP1A requires stimulation by the outer-membrane-anchored lipoprotein LpoA. Whereas the C-terminal domain of LpoA interacts with PBP1A to stimulate its peptide crosslinking activity, little is known about the role of the N-terminal domain. Herein we report its NMR structure, which adopts an all-α-helical fold comprising a series of helix-turn-helix tetratricopeptide-repeat (TPR)-like motifs. NMR spectroscopy of full-length LpoA revealed two extended flexible regions in the C-terminal domain and limited, if any, flexibility between the N- and C-terminal domains. Analytical ultracentrifugation and small-angle X-ray scattering results are consistent with LpoA adopting an elongated shape, with dimensions sufficient to span from the outer membrane through the periplasm to interact with the peptidoglycan synthase PBP1A. PMID:24954617

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation

    PubMed Central

    1996-01-01

    We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin- related proteins. Immunoblots using an antibody raised against a non- conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti-DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone. PMID:8636234

  2. 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.

  3. 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...

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation

    PubMed Central

    Deng, Ting; Yao, Hongyan; Wang, Jin; Wang, Jun; Xue, Hongwei; Zuo, Kaijing

    2016-01-01

    The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates. PMID:27311358

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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...

  7. 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...

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  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. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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)

  5. 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

  6. 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...

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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...

  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. 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. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. CsAGP1, a Gibberellin-Responsive Gene from Cucumber Hypocotyls, Encodes a Classical Arabinogalactan Protein and Is Involved in Stem Elongation

    PubMed Central

    Park, Me Hea; Suzuki, Yoshihito; Chono, Makiko; Knox, J. Paul; Yamaguchi, Isomaro

    2003-01-01

    Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a “classical” arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(βGlc)3-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(βGlc)3, which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation. PMID:12644694

  16. CsAGP1, a gibberellin-responsive gene from cucumber hypocotyls, encodes a classical arabinogalactan protein and is involved in stem elongation.

    PubMed

    Park, Me Hea; Suzuki, Yoshihito; Chono, Makiko; Knox, J Paul; Yamaguchi, Isomaro

    2003-03-01

    Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a "classical" arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(betaGlc)(3)-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(betaGlc)(3), which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation.

  17. 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

  18. 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

  19. 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.

  20. Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation.

    PubMed

    Kihara, Akio; Sakuraba, Hiroko; Ikeda, Mika; Denpoh, Aki; Igarashi, Yasuyuki

    2008-04-25

    Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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%.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Yin Yang 1: a multifaceted protein beyond a transcription factor.

    PubMed

    Deng, Zhiyong; Cao, Paul; Wan, Mei Mei; Sui, Guangchao

    2010-01-01

    As a transcription factor, Yin Yang 1 (YY1) regulates the transcription of a dazzling list of genes and the number of its targets still mounts. Recent studies revealed that YY1 possesses functions independent of its DNA binding activity and its regulatory role in tumorigenesis has started to emerge.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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...

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Elongation Factor-1a is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum*

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess a unique complex of organelles at the anterior end, referred to as the apical complex, which is involved in host cell invasion. Previously, we generated the chicken m...

  7. 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.

  8. 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.

  9. Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress

    PubMed Central

    Ameri, Kurosh; Rajah, Anthony M.; Nguyen, Vien; Sanders, Timothy A.; Jahangiri, Arman; DeLay, Michael; Donne, Matthew; Choi, Hwa J.; Tormos, Kathryn V.; Yeghiazarians, Yerem; Jeffrey, Stefanie S.; Rinaudo, Paolo F.; Rowitch, David H.; Aghi, Manish; Maltepe, Emin

    2013-01-01

    Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo. PMID:23646141

  10. 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...

  11. Plasma DYRK1A as a novel risk factor for Alzheimer's disease.

    PubMed

    Janel, N; Sarazin, M; Corlier, F; Corne, H; de Souza, L C; Hamelin, L; Aka, A; Lagarde, J; Blehaut, H; Hindié, V; Rain, J-C; Arbones, M L; Dubois, B; Potier, M C; Bottlaender, M; Delabar, J M

    2014-01-01

    To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-β42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD. PMID:25116835

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Cytochrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Hammond, Geoffrey L; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Breast cancer risk may be determined by various genetic, metabolic, and lifestyle factors that alter sex hormone metabolism. Cytochrome P450 1A2 (CYP1A2) is responsible for the metabolism of estrogens and many exogenous compounds, including caffeine. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity and known or suspected risk factors for breast cancer. Blood levels of sex hormones, lipids, and growth factors were measured. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Stepwise and maximum R regression analyses were used to identify covariates related to CYP1A2 activity after adjustment for ethnicity. Results In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche. Conclusion These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer. PMID:15217502

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.)

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Comparative genomic and functional analyses: unearthing the diversity and specificity of nematicidal factors in Pseudomonas putida strain 1A00316

    PubMed Central

    Guo, Jing; Jing, Xueping; Peng, Wen-Lei; Nie, Qiyu; Zhai, Yile; Shao, Zongze; Zheng, Longyu; Cai, Minmin; Li, Guangyu; Zuo, Huaiyu; Zhang, Zhitao; Wang, Rui-Ru; Huang, Dian; Cheng, Wanli; Yu, Ziniu; Chen, Ling-Ling; Zhang, Jibin

    2016-01-01

    We isolated Pseudomonas putida (P. putida) strain 1A00316 from Antarctica. This bacterium has a high efficiency against Meloidogyne incognita (M. incognita) in vitro and under greenhouse conditions. The complete genome of P. putida 1A00316 was sequenced using PacBio single molecule real-time (SMRT) technology. A comparative genomic analysis of 16 Pseudomonas strains revealed that although P. putida 1A00316 belonged to P. putida, it was phenotypically more similar to nematicidal Pseudomonas fluorescens (P. fluorescens) strains. We characterized the diversity and specificity of nematicidal factors in P. putida 1A00316 with comparative genomics and functional analysis, and found that P. putida 1A00316 has diverse nematicidal factors including protein alkaline metalloproteinase AprA and two secondary metabolites, hydrogen cyanide and cyclo-(l-isoleucyl-l-proline). We show for the first time that cyclo-(l-isoleucyl-l-proline) exhibit nematicidal activity in P. putida. Interestingly, our study had not detected common nematicidal factors such as 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin in P. putida 1A00316. The results of the present study reveal the diversity and specificity of nematicidal factors in P. putida strain 1A00316. PMID:27384076

  3. 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

  4. Aldehyde dehydrogenase 1A1 stabilizes transcription factor Gli2 and enhances the activity of Hedgehog signaling in hepatocellular cancer.

    PubMed

    Yan, Zhengwei; Xu, Liyao; Zhang, Junyan; Lu, Quqin; Luo, Shiwen; Xu, Linlin

    2016-03-18

    The Gli transcription factors are primary transcriptional regulators that mediate the activation of Hedgehog (Hh) signaling. Recent studies have revealed that Gli proteins are also regulated transcriptionally and post-translationally through noncanonical mechanisms, independent of Hh signaling. However, the precise mechanisms involved in the regulation of Gli proteins remain unclear. Using a differential mass-spectrometry approach, we found that aldehyde dehydrogenase 1A1 (ALDH1A1) is associated with transcription factor Gli2. Overexpression of ALDH1A1 increased Gli2 protein levels; in contrast, ALDH1A1 depletion facilitated Gli2 degradation. In addition, Gli2 mRNA expression was not affected by ectopic expression of ALDH1A1, indicating the role of ALDH1A1 in the stabilization of Gli2. Further investigation showed that ALDH1A1 prolonged the stability of Gli2 protein in a catalytic-independent manner. Finally, we showed that overexpression of ALDH1A1 activated the Hh signaling pathway and promoted cell growth, migration and invasion in hepatocellular cancer cells. Together, these results illustrate regulatory roles of ALDH1A1 in the activation of the Hh signaling pathway and highlight a novel mechanism for the aberrant activation of the Hh signaling pathway in hepatocellular cancer cells. PMID:26896768

  5. Up-regulation of CHAF1A, a poor prognostic factor, facilitates cell proliferation of colon cancer

    SciTech Connect

    Wu, Zehua; Cui, Feifei; Yu, Fudong; Peng, Xiao; Jiang, Tao; Chen, Dawei; Lu, Su; Tang, Huamei; Peng, Zhihai

    2014-06-27

    Highlights: • We identified that CHAF1A was up-regulated in colon tumor mucosa in TMA. • The expression pattern of CHAF1A was validated with qPCR and western-blot. • CHAF1A overexpression is an independent indicator for poor colon cancer survival. • CHAF1A facilitates cell proliferation of colon cancer both in vitro and in vivo. - Abstract: Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.

  6. Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A.

    PubMed Central

    Kraus, V B; Inostroza, J A; Yeung, K; Reinberg, D; Nevins, J R

    1994-01-01

    Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex. Images PMID:8022773

  7. Hepatocyte Nuclear Factor 1A Is a Cell-Intrinsic Transcription Factor Required for B Cell Differentiation and Development in Mice.

    PubMed

    von Wnuck Lipinski, Karin; Sattler, Katherine; Peters, Susann; Weske, Sarah; Keul, Petra; Klump, Hannes; Heusch, Gerd; Göthert, Joachim R; Levkau, Bodo

    2016-02-15

    The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A(-/-) mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A(-/-) bone marrow, all B cell progenitors-from pre-pro-/early pro-B cells to immature B cells-were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A(-/-) mice, whereas IL-7 stimulation of HNF1A(-/-) B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A(-/-) common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A(-/-) bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A(-/-) lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.

  8. The multifunctional transcription factor Rap1: a regulator of yeast physiology.

    PubMed

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-01-01

    Transcription is a fundamental process that is tightly regulated by transcription factors to maintain cellular homeostasis. Transcription factors have DNA-binding domains, some of which are sequence specific, and are found throughout the eukaryotic kingdom. Recent studies have revealed the molecular mechanisms by which transcription factors perform their functions. In the budding yeast Saccharomyces cerevisiae, Rap1 (ScRap1) can either activate or repress transcription. This bimodal transcriptional activity has led to the widespread study of the mode of action of ScRap1. This review summarizes current knowledge about yeast ScRap1, including its structure, mechanisms of transcription regulation, and biological functions, and the future directions in the field.

  9. [Trefoil factor 1 (pS2/TFF1), a peptide with numerous functions].

    PubMed

    Mathelin, Carole; Tomasetto, Catherine; Rio, Marie-Christine

    2005-09-01

    The trefoil factor (TFF) family includes three members : TFF1 also called pS2, TFF2 or spasmolytic peptide (SP) and TFF3 or intestinal trefoil factor (ITF). TFFs are associated with mucin-secreting epithelial cells and play a crucial role in mucosal defense and healing. In case of mucosa aggression (due to bacteria, virus or medication), inflammatory diseases and ulcerous pathology, they are involved in epithelium restitution and regeneration. In comparison with the other TFFs, pS2/TFF1 has particular functions. Notably, it acts like a suppressor gene for gastric tumors. Evidence of pS2/TFF1 overexpression has been found in a range of carcinomas (breast, bowel, prostate, pancreas, thyroid, lung...). In breast cancer, pS2/TFF1 overexpression contributes to a favorable prognosis. Moreover, it is a predictive factor of hormonotherapy response.

  10. Adenovirus E1A Targets the DREF Nuclear Factor To Regulate Virus Gene Expression, DNA Replication, and Growth

    PubMed Central

    Radko, Sandi; Koleva, Maria; James, Kris M. D.; Jung, Richard; Mymryk, Joe S.

    2014-01-01

    ABSTRACT The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication. IMPORTANCE This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to

  11. Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage.

    PubMed

    Her, Joonyoung; Soo Lee, Nam; Kim, Yonghwan; Kim, Hongtae

    2016-07-01

    Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex.

  12. Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage.

    PubMed

    Her, Joonyoung; Soo Lee, Nam; Kim, Yonghwan; Kim, Hongtae

    2016-07-01

    Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex. PMID:27325824

  13. The transcription factor Egr-1: a potential drug in wound healing and tissue repair.

    PubMed

    Braddock, M

    2001-07-01

    In the United States, between 40 and 90 million hospital days are lost per year as a result of trauma and surgical procedures which result in the loss of functional tissue. This is estimated to cost the economy and healthcare providers in excess of US$ 500 billion, a figure that is increasing because of extending population lifespan. Tissue engineering and gene therapies are radical new treatments that are aimed at tissue regeneration ranging from dermal, osteal and occular repair to the replacement of failing tissue with entire biosynthetic organs. Over the last decade, numerous proteins have been identified that are able to direct the synthesis of new tissue. Such proteins include growth factors, cytokines and, more recently, transcription factors.

  14. Human Sos1: A guanine nucleotide exchange factor for ras that binds to GRB2

    SciTech Connect

    Chardin, P. ); Camonis, J.; Gale, N.W.; Aelst, L. Van; Wigler, M.H.; Bar-Sagi, D. ); Schlessinger, J. )

    1993-05-28

    A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1. 42 refs., 5 figs.

  15. Influence of environmental and genetic factors on CYP1A2 activity in individuals of South Asian and European ancestry.

    PubMed

    Perera, V; Gross, A S; McLachlan, A J

    2012-10-01

    The drug-metabolizing enzyme CYP1A2 contributes to the metabolism of a number of commonly used medicines and displays wide interindividual variability. The aim of this study was to investigate CYP1A2 activity in a population of South Asian ancestry and compare it with a population of European ancestry. CYP1A2 activity was determined using the 4 h paraxanthine/caffeine saliva concentration ratio following a 100-mg oral dose of caffeine in healthy individuals of South Asian (n = 166) and European (n = 166) ancestry. Participants were surveyed for extrinsic ethnic factors and genotyped for polymorphisms in CYP1A2 and related genes. Significantly lower CYP1A2 activity was observed in South Asian participants (median: 0.42; range: 0.10-1.06) as compared with European participants (0.54; 0.12-1.64) (P < 0.01). Multiple linear regression indicated that 41% of the variability in CYP1A2 activity could be explained by the diet, lifestyle, and genetic factors studied.

  16. Causes of Death and Prognostic Factors in Multiple Endocrine Neoplasia Type 1: A Prospective Study

    PubMed Central

    Ito, Tetsuhide; Igarashi, Hisato; Uehara, Hirotsugu; Berna, Marc J.; Jensen, Robert T.

    2013-01-01

    Abstract Multiple endocrine neoplasia type 1 (MEN1) is classically characterized by the development of functional or nonfunctional hyperplasia or tumors in endocrine tissues (parathyroid, pancreas, pituitary, adrenal). Because effective treatments have been developed for the hormone excess state, which was a major cause of death in these patients in the past, coupled with the recognition that nonendocrine tumors increasingly develop late in the disease course, the natural history of the disease has changed. An understanding of the current causes of death is important to tailor treatment for these patients and to help identify prognostic factors; however, it is generally lacking. To add to our understanding, we conducted a detailed analysis of the causes of death and prognostic factors from a prospective long-term National Institutes of Health (NIH) study of 106 MEN1 patients with pancreatic endocrine tumors with Zollinger-Ellison syndrome (MEN1/ZES patients) and compared our results to those from the pooled literature data of 227 patients with MEN1 with pancreatic endocrine tumors (MEN1/PET patients) reported in case reports or small series, and to 1386 patients reported in large MEN1 literature series. In the NIH series over a mean follow-up of 24.5 years, 24 (23%) patients died (14 MEN1-related and 10 non-MEN1-related deaths). Comparing the causes of death with the results from the 227 patients in the pooled literature series, we found that no patients died of acute complications due to acid hypersecretion, and 8%–14% died of other hormone excess causes, which is similar to the results in 10 large MEN1 literature series published since 1995. In the 2 series (the NIH and pooled literature series), two-thirds of patients died from an MEN1-related cause and one-third from a non-MEN1-related cause, which agrees with the mean values reported in 10 large MEN1 series in the literature, although in the literature the causes of death varied widely. In the NIH and pooled

  17. AKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

    PubMed Central

    Burla, Romina; Carcuro, Mariateresa; Raffa, Grazia D.; Galati, Alessandra; Raimondo, Domenico; Rizzo, Angela; La Torre, Mattia; Micheli, Emanuela; Ciapponi, Laura; Cenci, Giovanni; Cundari, Enrico; Musio, Antonio; Biroccio, Annamaria; Cacchione, Stefano; Gatti, Maurizio; Saggio, Isabella

    2015-01-01

    Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively

  18. CFL1, a WW Domain Protein, Regulates Cuticle Development by Modulating the Function of HDG1, a Class IV Homeodomain Transcription Factor, in Rice and Arabidopsis[W

    PubMed Central

    Wu, Renhong; Li, Shibai; He, Shan; Waßmann, Friedrich; Yu, Caihong; Qin, Genji; Schreiber, Lukas; Qu, Li-Jia; Gu, Hongya

    2011-01-01

    Plants have a chemically heterogeneous lipophilic layer, the cuticle, which protects them from biotic and abiotic stresses. The mechanisms that regulate cuticle development are poorly understood. We identified a rice (Oryza sativa) dominant curly leaf mutant, curly flag leaf1 (cfl1), and cloned CFL1, which encodes a WW domain protein. We overexpressed both rice and Arabidopsis CFL1 in Arabidopsis thaliana; these transgenic plants showed severely impaired cuticle development, similar to that in cfl1 rice. Reduced expression of At CFL1 resulted in reinforcement of cuticle structure. At CFL1 was predominantly expressed in specialized epidermal cells and in regions where dehiscence and abscission occur. Biochemical evidence showed that At CFL1 interacts with HDG1, a class IV homeodomain-leucine zipper transcription factor. Suppression of HDG1 function resulted in similar defective cuticle phenotypes in wild-type Arabidopsis but much alleviated phenotypes in At cfl1-1 mutants. The expression of two cuticle development-associated genes, BDG and FDH, was downregulated in At CFL1 overexpressor and HDG1 suppression plants. HDG1 binds to the cis-element L1 box, which exists in the regulatory regions of BDG and FDH. Our results suggest that rice and Arabidopsis CFL1 negatively regulate cuticle development by affecting the function of HDG1, which regulates the downstream genes BDG and FDH. PMID:21954461

  19. 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

  20. 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.

  1. Lipase maturation factor 1: a lipase chaperone involved in lipid metabolism.

    PubMed

    Péterfy, Miklós

    2012-05-01

    Mutations in lipase maturation factor 1 (LMF1) are associated with severe hypertriglyceridemia in mice and human subjects. The underlying cause is impaired lipid clearance due to lipase deficiency. LMF1 is a chaperone of the endoplasmic reticulum (ER) and it is critically required for the post-translational activation of three vascular lipases: lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). As LMF1 is only required for the maturation of homodimeric, but not monomeric, lipases, it is likely involved in the assembly of inactive lipase subunits into active enzymes and/or the stabilization of active dimers. Herein, we provide an overview of current understanding of LMF1 function and propose that it may play a regulatory role in lipase activation and lipid metabolism. Further studies will be required to test this hypothesis and elucidate the full spectrum of phenotypes in combined lipase deficiency. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease. PMID:22063272

  2. 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.

  3. Interactions between Environmental Factors and Melatonin Receptor Type 1A Polymorphism in Relation to Oral Cancer Susceptibility and Clinicopathologic Development

    PubMed Central

    Yang, Shun-Fa; Lee, Wei-Jiunn; Lin, Yung-Wei; Lee, Liang-Ming; Chang, Junn-Liang; Weng, Wei-Chun; Lin, Chien-Huang; Chien, Ming-Hsien

    2015-01-01

    Background The purpose of this study was to explore the combined effect of melatonin receptor type 1A (MTNR1A) gene polymorphisms and exposure to environmental carcinogens on the susceptibility and clinicopathological characteristics of oral cancer. Methodology and Principal Findings Three polymorphisms of the MTNR1A gene from 618 patients with oral cancer and 560 non-cancer controls were analyzed by real-time polymerase chain reaction (PCR). The CTA haplotype of the studied MTNR1A polymorphisms (rs2119882, rs13140012, rs6553010) was related to a higher risk of oral cancer. Moreover, MTNR1A gene polymorphisms exhibited synergistic effects of environmental factors (betel quid and tobacco use) on the susceptibility of oral cancer. Finally, oral-cancer patients with betel quid-chewing habit who had T/T allele of MTNR1A rs13140012 were at higher risk for developing an advanced clinical stage and lymph node metastasis. Conclusion These results support gene-environment interactions of MTNR1A polymorphisms with smoking and betel quid-chewing habits possibly altering oral-cancer susceptibility and metastasis. PMID:25806809

  4. Angiotensin type 1a receptors on corticotropin-releasing factor neurons contribute to the expression of conditioned fear.

    PubMed

    Hurt, R C; Garrett, J C; Keifer, O P; Linares, A; Couling, L; Speth, R C; Ressler, K J; Marvar, P J

    2015-09-01

    Although generally associated with cardiovascular regulation, angiotensin II receptor type 1a (AT1a R) blockade in mouse models and humans has also been associated with enhanced fear extinction and decreased post-traumatic stress disorder (PTSD) symptom severity, respectively. The mechanisms mediating these effects remain unknown, but may involve alterations in the activities of corticotropin-releasing factor (CRF)-expressing cells, which are known to be involved in fear regulation. To test the hypothesis that AT1a R signaling in CRFergic neurons is involved in conditioned fear expression, we generated and characterized a conditional knockout mouse strain with a deletion of the AT1a R gene from its CRF-releasing cells (CRF-AT1a R((-/-)) ). These mice exhibit normal baseline heart rate, blood pressure, anxiety and locomotion, and freeze at normal levels during acquisition of auditory fear conditioning. However, CRF-AT1a R((-/-)) mice exhibit less freezing than wild-type mice during tests of conditioned fear expression-an effect that may be caused by a decrease in the consolidation of fear memory. These results suggest that central AT1a R activity in CRF-expressing cells plays a role in the expression of conditioned fear, and identify CRFergic cells as a population on which AT1 R antagonists may act to modulate fear extinction.

  5. 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.

  6. Immune factor Gambif1, a new rel family member from the human malaria vector, Anopheles gambiae.

    PubMed Central

    Barillas-Mury, C; Charlesworth, A; Gross, I; Richman, A; Hoffmann, J A; Kafatos, F C

    1996-01-01

    A novel rel family member, Gambif1 (gambiae immune factor 1), has been cloned from the human malaria vector, Anopheles gambiae, and shown to be most similar to Drosophila Dorsal and Dif. Gambif1 protein is translocated to the nucleus in fat body cells in response to bacterial challenge, although the mRNA is present at low levels at all developmental stages and is not induced by infection. DNA binding activity to the kappaB-like sites in the A.gambiae Defensin and the Drosophila Diptericin and Cecropin promoters is also induced in larval nuclear extracts following infection. Gambif1 has the ability to bind to kappaB-like sites in vitro. Co-transfection assays in Drosophila mbn-2 cells show that Gambif1 can activate transcription by interacting with the Drosophila Diptericin regulatory elements, but is not functionally equivalent to Dorsal in this assay. Gambif1 protein translocation to the nucleus and the appearance of kappaB-like DNA binding activity can serve as molecular markers of activation of the immune system and open up the possibility of studying the role of defence reactions in determining mosquito susceptibility/refractoriness to malaria infection. Images PMID:8887560

  7. Hypoxia-Inducible Factor-1 (HIF-1): A Potential Target for Intervention in Ocular Neovascular Diseases

    PubMed Central

    Vadlapatla, Ramya Krishna; Vadlapudi, Aswani Dutt; Mitra, Ashim K.

    2015-01-01

    Constant oxygen supply is essential for proper tissue development, homeostasis and function of all eukaryotic organisms. Cellular response to reduced oxygen levels is mediated by the transcriptional regulator hypoxia-inducible factor-1 (HIF-1). It is a heterodimeric complex protein consisting of an oxygen dependent subunit (HIF-1α) and a constitutively expressed nuclear subunit (HIF-1β). In normoxic conditions, de novo synthesized cytoplasmic HIF-1α is degraded by 26S proteasome. Under hypoxic conditions, HIF-1α is stabilized, binds with HIF-1β and activates transcription of various target genes. These genes play a key role in regulating angiogenesis, cell survival, proliferation, chemotherapy, radiation resistance, invasion, metastasis, genetic instability, immortalization, immune evasion, metabolism and stem cell maintenance. This review highlights the importance of hypoxia signaling in development and progression of various vision threatening pathologies such as diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration and glaucoma. Further, various inhibitors of HIF-1 pathway that may have a viable potential in the treatment of oxygen-dependent ocular diseases are also discussed. PMID:23701276

  8. Do psychological factors influence recovery from complex regional pain syndrome type 1? A prospective study.

    PubMed

    Bean, Debbie J; Johnson, Malcolm H; Heiss-Dunlop, Wolfgang; Lee, Arier C; Kydd, Robert R

    2015-11-01

    Previous studies have shown that the outcomes of complex regional pain syndrome (CRPS) vary significantly between patients, but few studies have identified prognostic indicators. The aim of this study was to determine whether psychological factors are associated with recovery from recently onset CRPS amongst patients followed prospectively for 1 year. Sixty-six patients with CRPS (type 1) were recruited within 12 weeks of symptom onset and assessed immediately and at 6 and 12 months, during which time they received treatment as usual. At each assessment, the following were measured: signs and symptoms of CRPS, pain, disability, depression, anxiety, stress, pain-related fear, pain catastrophising, laterality task performance, body perception disturbance, and perceived ownership of the limb. Mixed-effects models for repeated measures were conducted to identify baseline variables associated with CRPS severity, pain, and disability over the 12 months. Results showed that scores for all 3 outcome variables improved over the study period. Males and those with lower levels of baseline pain and disability experienced the lowest CRPS severity scores over 12 months. Those with lower baseline anxiety and disability had the lowest pain intensity over the study period, and those with lower baseline pain and pain-related fear experienced the least disability over the 12 months. This suggests that anxiety, pain-related fear, and disability are associated with poorer outcomes in CRPS and could be considered as target variables for early treatment. The findings support the theory that CRPS represents an aberrant protective response to perceived threat of tissue injury.

  9. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor.

    PubMed

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés; Barrera-Figueroa, Blanca Estela; Peña-Castro, Julián Mario

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200-300% more glucose, adult vegetative plants yielded 40-90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production.

  10. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

    PubMed Central

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production. PMID:25780769

  11. The adenine nucleotide translocase type 1 (ANT1): a new factor in mitochondrial disease.

    PubMed

    Sharer, J Daniel

    2005-09-01

    Mitochondrial disorders of oxidative phosphorylation (OXPHOS) comprise a growing list of potentially lethal diseases caused by mutations in either mitochondrial (mtDNA) or nuclear DNA (nDNA). Two such conditions, autosomal dominant progressive external ophthalmoplegia (adPEO) and Senger's Syndrome, are associated with dysfunction of the heart and muscle-specific isoform of the adenine nucleotide translocase (ANT1), a nDNA gene product that facilitates transport of ATP and ADP across the inner mitochondrial membrane. AdPEO is a mtDNA deletion disorder broadly characterized by pathology involving the eyes, skeletal muscle, and central nervous system. In addition to ANT1, mutations in at least two other nuclear genes, twinkle and POLG, have been shown to cause mtDNA destabilization associated with adPEO. Senger's syndrome is an autosomal recessive condition characterized by congenital heart defects, abnormalities of skeletal muscle mitochondria, cataracts, and elevated circulatory levels of lactic acid. This syndrome is associated with severe depletion of ANT1, which may be the result of an as yet unidentified ANT1-specific transcriptional or translational processing error. ANT1 has also been associated with a third condition, autosomal dominant facioscapulohumeral muscular dystrophy (FSHD), an adult onset disorder characterized by variable muscle weakness in the face, feet, shoulders, and hips. FSHD patients possess specific DNA deletions on chromosome 4, which appear to cause derepression of several nearby genes, including ANT1. Early development of FSHD may involve mitochondrial dysfunction and increased oxidative stress, possibly associated with overexpression of ANT1. PMID:16203679

  12. Molecular characterization of Quercus suber MYB1, a transcription factor up-regulated in cork tissues.

    PubMed

    Almeida, Tânia; Menéndez, Esther; Capote, Tiago; Ribeiro, Teresa; Santos, Conceição; Gonçalves, Sónia

    2013-01-15

    The molecular processes associated with cork development in Quercus suber L. are poorly understood. A previous molecular approach identified a list of genes potentially important for cork formation and differentiation, providing a new basis for further molecular studies. This report is the first molecular characterization of one of these candidate genes, QsMYB1, coding for an R2R3-MYB transcription factor. The R2R3-MYB gene sub-family has been described as being involved in the phenylpropanoid and lignin pathways, both involved in cork biosynthesis. The results showed that the expression of QsMYB1 is putatively mediated by an alternative splicing (AS) mechanism that originates two different transcripts (QsMYB1.1 and QsMYB1.2), differing only in the 5'-untranslated region, due to retention of the first intron in one of the variants. Moreover, within the retained intron, a simple sequence repeat (SSR) was identified. The upstream regulatory region of QsMYB1 was extended by a genome walking approach, which allowed the identification of the putative gene promoter region. The relative expression pattern of QsMYB1 transcripts determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that both transcripts were up-regulated in cork tissues; the detected expression was several times higher in newly formed cork harvested from trees producing virgin, second or reproduction cork when compared with wood. Moreover, the expression analysis of QsMYB1 in several Q. suber organs showed very low expression in young branches and roots, whereas in leaves, immature acorns or male flowers, no expression was detected. These preliminary results suggest that QsMYB1 may be related to secondary growth and, in particular, with the cork biosynthesis process with a possible alternative splicing mechanism associated with its regulatory function.

  13. Do psychological factors influence recovery from complex regional pain syndrome type 1? A prospective study.

    PubMed

    Bean, Debbie J; Johnson, Malcolm H; Heiss-Dunlop, Wolfgang; Lee, Arier C; Kydd, Robert R

    2015-11-01

    Previous studies have shown that the outcomes of complex regional pain syndrome (CRPS) vary significantly between patients, but few studies have identified prognostic indicators. The aim of this study was to determine whether psychological factors are associated with recovery from recently onset CRPS amongst patients followed prospectively for 1 year. Sixty-six patients with CRPS (type 1) were recruited within 12 weeks of symptom onset and assessed immediately and at 6 and 12 months, during which time they received treatment as usual. At each assessment, the following were measured: signs and symptoms of CRPS, pain, disability, depression, anxiety, stress, pain-related fear, pain catastrophising, laterality task performance, body perception disturbance, and perceived ownership of the limb. Mixed-effects models for repeated measures were conducted to identify baseline variables associated with CRPS severity, pain, and disability over the 12 months. Results showed that scores for all 3 outcome variables improved over the study period. Males and those with lower levels of baseline pain and disability experienced the lowest CRPS severity scores over 12 months. Those with lower baseline anxiety and disability had the lowest pain intensity over the study period, and those with lower baseline pain and pain-related fear experienced the least disability over the 12 months. This suggests that anxiety, pain-related fear, and disability are associated with poorer outcomes in CRPS and could be considered as target variables for early treatment. The findings support the theory that CRPS represents an aberrant protective response to perceived threat of tissue injury. PMID:26133727

  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. Carnitine palmitoyltransferase 1A functions to repress FoxO transcription factors to allow cell cycle progression in ovarian cancer

    PubMed Central

    Shao, Huanjie; Mohamed, Esraa M.; Xu, Guoyan G.; Waters, Michael; Jing, Kai; Ma, Yibao; Zhang, Yan; Spiegel, Sarah; Idowu, Michael O.; Fang, Xianjun

    2016-01-01

    Cancer cells rely on hyperactive de novo lipid synthesis for maintaining malignancy. Recent studies suggest involvement in cancer of fatty acid oxidation, a process functionally opposite to lipogenesis. A mechanistic link from lipid catabolism to oncogenic processes is yet to be established. Carnitine palmitoyltransferase 1 (CPT1) is a rate-limiting enzyme of fatty acid β-oxidation (FAO) that catalyzes the transfer of long-chain acyl group of the acyl-CoA ester to carnitine, thereby shuttling fatty acids into the mitochondrial matrix for β-oxidation. In the present study, we demonstrated that CPT1A was highly expressed in most ovarian cancer cell lines and primary ovarian serous carcinomas. Overexpression of CPT1A correlated with a poor overall survival of ovarian cancer patients. Inactivation of CPT1A decreased cellular ATP levels and induced cell cycle arrest at G0/G1, suggesting that ovarian cancer cells depend on or are addicted to CPT1A-mediated FAO for cell cycle progression. CPT1A deficiency also suppressed anchorage-independent growth and formation of xenografts from ovarian cancer cell lines. The cyclin-dependent kinase inhibitor p21WAF1 (p21) was identified as most consistently and robustly induced cell cycle regulator upon inactivation of CPT1A. Furthermore, p21 was transcriptionally upregulated by the FoxO transcription factors, which were in turn phosphorylated and activated by AMP-activated protein kinase and the mitogen-activated protein kinases JNK and p38. Our results established the oncogenic relevance of CPT1A and a mechanistic link from lipid catabolism to cell cycle regulation, suggesting that CPT1A could be a prognostic biomarker and rational target for therapeutic intervention of cancer. PMID:26716645

  16. Increased serotonin-1A (5-HT1A) autoreceptor expression and reduced raphe serotonin levels in deformed epidermal autoregulatory factor-1 (Deaf-1) gene knock-out mice.

    PubMed

    Czesak, Margaret; Le François, Brice; Millar, Anne M; Deria, Mariam; Daigle, Mireille; Visvader, Jane E; Anisman, Hymie; Albert, Paul R

    2012-02-24

    Altered regulation of the serotonin-1A (5-HT1A) receptor gene is implicated in major depression and mood disorders. The functional human 5-HT1A C(-1019)G promoter polymorphism (rs6295), which prevents the binding of Deaf-1/NUDR leading to dysregulation of the receptor, has been associated with major depression. In cell models Deaf-1 displays dual activity, repressing 5-HT1A autoreceptor expression in serotonergic raphe cells while enhancing postsynaptic 5-HT1A heteroreceptor expression in nonserotonergic neurons. A functional Deaf-1 binding site on the mouse 5-HT1A promoter was recognized by Deaf-1 in vitro and in vivo and mediated dual activity of Deaf-1 on 5-HT1A gene transcription. To address regulation by Deaf-1 in vivo, Deaf-1 knock-out mice bred to a C57BL/6 background were compared with wild-type siblings for changes in 5-HT1A RNA and protein by quantitative RT-PCR, in situ hybridization, and immunofluorescence. In the dorsal raphe, Deaf-1 knock-out mice displayed increased 5-HT1A mRNA, protein, and 5-HT1A-positive cell counts but reduced 5-HT levels, whereas other serotonergic markers, such as tryptophan hydroxylase (TPH)- or 5-HT-positive cells and TPH2 RNA levels, were unchanged. By contrast, 5-HT1A mRNA and 5-HT1A-positive cells were reduced in the frontal cortex of Deaf-1-null mice, with no significant change in hippocampal 5-HT1A RNA, protein, or cell counts. The region-specific alterations of brain 5-HT1A gene expression and reduced raphe 5-HT content in Deaf-1(-/-) mice indicate the importance of Deaf-1 in regulation of 5-HT1A gene expression and provide insight into the role of the 5-HT1A G(-1019) allele in reducing serotonergic neurotransmission by derepression of 5-HT1A autoreceptors.

  17. Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

    PubMed

    Pessler, F; Pendergrast, P S; Hernandez, N

    1997-07-01

    The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

  18. Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

    PubMed Central

    Pessler, F; Pendergrast, P S; Hernandez, N

    1997-01-01

    The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes. PMID:9199312

  19. 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.

  20. On the role of 5-HT(1A) receptor gene in behavioral effect of brain-derived neurotrophic factor.

    PubMed

    Naumenko, Vladimir S; Kondaurova, Elena M; Bazovkina, Daria V; Tsybko, Anton S; Il'chibaeva, Tatyana V; Popova, Nina K

    2014-08-01

    Experiments were made on a congenic AKR.CBA-D13Mit76C (76C) mouse strain created by transferring a chromosome 13 fragment containing the 5-HT1A receptor gene from a CBA strain to an AKR background. It was shown that 76C mice differed from AKR mice by decreased 5-HT1A receptor and tryptophan hydroxylase-2 (tph-2) genes expression in the midbrain. Functional activity of 5-HT2A receptors and 5-HT(2A) receptor mRNA levels in the midbrain and hippocampus of 76C mice were decreased compared with AKR mice. Central brain-derived neurotrophic factor (BDNF) administration (300 ng i.c.v.) reduced 5-HT1A and 5-HT(2A) receptor mRNA levels in the frontal cortex and tph-2 mRNA level in the midbrain of AKR mice. However, BDNF failed to produce any effect on the expression of 5-HT(1A) , 5-HT(2A) , and tph-2 genes in 76C mice but decreased functional activity of 5-HT(2A) receptors in 76C mice and increased it in AKR mice. BDNF restored social deficiency in 76C mice but produced asocial behavior (aggressive attacks towards young mice) in AKR mice. The data indicate that a small genetic variation altered the response to BDNF and show an important role of 5-HT(1A) receptor gene in the 5-HT system response to BDNF treatment and in behavioral effects of BDNF.

  1. Angiotensin Type 1a Receptors on Corticotropin-Releasing Factor Neurons Contribute to the Expression of Conditioned Fear

    PubMed Central

    Hurt, Robert C.; Garrett, Jacob C.; Keifer, Orion P.; Linares, Andrea; Couling, Leena; Speth, Robert C.; Ressler, Kerry J.; Marvar, Paul J.

    2015-01-01

    Although generally associated with cardiovascular regulation, angiotensin II receptor type 1 (AT1aR) blockade in mouse models and humans has also been associated with enhanced fear extinction and decreased post-traumatic stress disorder (PTSD) symptom severity, respectively. The mechanisms mediating these effects remain unknown, but may involve alterations in the activities of corticotropin-releasing factor (CRF)-expressing cells, which are known to be involved in fear regulation. To test the hypothesis that AT1aR signaling in CRFergic neurons is involved in conditioned fear expression, we generated and characterized a conditional knockout mouse strain with a deletion of the AT1aR gene from its CRF-releasing cells (CRF-AT1aR(−/−)). These mice exhibit normal baseline heart rate, blood pressure, anxiety, and locomotion, and freeze at normal levels during acquisition of auditory fear conditioning. However, CRF-AT1aR(−/−) mice exhibit less freezing than wild type mice during tests of conditioned fear expression—an effect that may be caused by a decrease in the consolidation of fear memory. These results suggest that central AT1R activity in CRF-expressing cells plays a role in the expression of conditioned fear, and identify CRFergic cells as a population on which AT1R antagonists may act to modulate fear extinction. PMID:26257395

  2. Ectopic Expression of DREB Transcription Factor, AtDREB1A, Confers Tolerance to Drought in Transgenic Salvia miltiorrhiza.

    PubMed

    Wei, Tao; Deng, Kejun; Liu, Dongqing; Gao, Yonghong; Liu, Yu; Yang, Meiling; Zhang, Lipeng; Zheng, Xuelian; Wang, Chunguo; Song, Wenqin; Chen, Chengbin; Zhang, Yong

    2016-08-01

    Drought decreases crop productivity more than any other type of environmental stress. Transcription factors (TFs) play crucial roles in regulating plant abiotic stress responses. The Arabidopsis thaliana gene DREB1A/CBF3, encoding a stress-inducible TF, was introduced into Salvia miltiorrhiza Ectopic expression of AtDREB1A resulted in increased drought tolerance, and transgenic lines had higher relative water content and Chl content, and exhibited an increased photosynthetic rate when subjected to drought stress. AtDREB1A transgenic plants generally displayed lower malondialdehyde (MDA), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress. In particular, plants with ectopic AtDREB1A expression under the control of the stress-induced RD29A promoter exhibited more tolerance to drought compared with p35S::AtDREB1A transgenic plants, without growth inhibition or phenotypic aberrations. Differential gene expression profiling of wild-type and pRD29A::AtDREB1A transgenic plants following drought stress revealed that the expression levels of various genes associated with the stress response, photosynthesis, signaling, carbohydrate metabolism and protein protection were substantially higher in transgenic plants. In addition, the amount of salvianolic acids and tanshinones was significantly elevated in AtDREB1A transgenic S. miltiorrhiza roots, and most of the genes in the related biosynthetic pathways were up-regulated. Together, these results demonstrated that inducing the expression of a TF can effectively regulate multiple genes in the stress response pathways and significantly improve the resistance of plants to abiotic stresses. Our results also suggest that genetic manipulation of a TF can improve production of valuable secondary metabolites by regulating genes in associated pathways. PMID:27485523

  3. 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

  4. 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

  5. Increased dosage of Dyrk1A alters alternative splicing factor (ASF)-regulated alternative splicing of tau in Down syndrome.

    PubMed

    Shi, Jianhua; Zhang, Tianyi; Zhou, Chunlei; Chohan, Muhammad Omar; Gu, Xiaosong; Wegiel, Jerzy; Zhou, Jianhua; Hwang, Yu-Wen; Iqbal, Khalid; Grundke-Iqbal, Inge; Gong, Cheng-Xin; Liu, Fei

    2008-10-17

    Two groups of tau, 3R- and 4R-tau, are generated by alternative splicing of tau exon 10. Normal adult human brain expresses equal levels of them. Disruption of the physiological balance is a common feature of several tauopathies. Very early in their life, individuals with Down syndrome (DS) develop Alzheimer-type tau pathology, the molecular basis for which is not fully understood. Here, we demonstrate that Dyrk1A, a kinase encoded by a gene in the DS critical region, phosphorylates alternative splicing factor (ASF) at Ser-227, Ser-234, and Ser-238, driving it into nuclear speckles and preventing it from facilitating tau exon 10 inclusion. The increased dosage of Dyrk1A in DS brain due to trisomy of chromosome 21 correlates to an increase in 3R-tau level, which on abnormal hyperphosphorylation and aggregation of tau results in neurofibrillary degeneration. Imbalance of 3R- and 4R-tau in DS brain by Dyrk1A-induced dysregulation of alternative splicing factor-mediated alternative splicing of tau exon 10 represents a novel mechanism of neurofibrillary degeneration and may help explain early onset tauopathy in individuals with DS.

  6. 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

  7. 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

  8. Peroxiredoxin-mediated redox regulation of the nuclear localization of Yap1, a transcription factor in budding yeast.

    PubMed

    Okazaki, Shoko; Naganuma, Akira; Kuge, Shusuke

    2005-01-01

    A redox reaction involving cysteine thiol-disulfide exchange is crucial for the intracellular monitoring of oxidation status. The yeast transcription factor Yap1 is activated by formation of a disulfide bond, which inhibits nuclear export in response to peroxide stress, with resultant enhancement of the nuclear localization of Yap1. A glutathione peroxidase-like protein, Gpx3, which has peroxiredoxin activity, is required for formation of the disulfide bond in Yap1. We show here that the requirement for Gpx3 in the regulation of Yap1 is strain-specific. Thus, Tsa1, a ubiquitous thioredoxin peroxidase, is required for the activation of Yap1 in yeast strain Y700, which is derived from W303. The strain-specific utilization of different peroxiredoxins appears to be determined by Ybp1, a Yap1-binding protein. The Ybp1 of Y700 has a nonsense mutation, and a wild-type YBP1 gene can restore the Gpx3-dependent activation of Yap1. These results suggest that Tsa1, a ubiquitous peroxiredoxin, has the potential for transducing redox signals to a particular sensor protein. PMID:15706081

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. Extracellular heat shock protein 70 (HSPA1A) and classical vascular risk factors in a general population

    PubMed Central

    Dulin, Elena; García-Barreno, Pedro

    2010-01-01

    Atherosclerosis is a chronic inflammatory and autoimmune disease. Candidate molecules/autoantigens include heat shock proteins (HSPs); Hsp70 (HSPA1A) is one of the best studied HSPs. Various studies have shown a correlation between extracellular Hsp70 (eHsp70) and anti-Hsp70/anti-Hsp60 antibody concentration and development of atherosclerosis. A random sample of 456 people aged 40–60 (218 males, 234 females) was studied to investigate the prevalence of traditional vascular risk factors and eHsp70 and anti-Hsp70/anti-Hsp60 antibodies levels, according to the risk of vascular disease. Task Force Chart was applied for classification. Subjects were divided into three groups: G0 (with no vascular risk factor or a risk lower than 5%), n = 239; G1 (moderated 10–20% risk, who do not have established disease) n = 161; and G2 (established atherosclerosis disease) n = 52. eHsp70 and anti-Hsp70 were significantly lower in the atherosclerosis group (group 2) with respect to the other groups. Disease-free people showed the highest anti-Hsp60 concentration compared with the other two groups. A correlation has not been demonstrated between the concentrations of circulating Hsp70 (HSPA1A), anti-Hsp70, and anti-Hsp60 and classical vascular risk factors and C-reactive protein. Low levels of eHsp70 and anti-Hsp70 antibodies should be considered as candidate FRV. Simultaneous decrease of eHsp70 and anti-Hsp70 antibodies would be explained by circulating immune complex formation, and both could be proposed as biomarkers for the progression of atherosclerotic disease. Levels of circulating anti-Hsp60 antibodies may constitute a marker of inflammation in atherosclerosis. PMID:20490736

  17. BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei.

    PubMed

    Vélez-Ramírez, D E; Florencio-Martínez, L E; Romero-Meza, G; Rojas-Sánchez, S; Moreno-Campos, R; Arroyo, R; Ortega-López, J; Manning-Cela, R; Martínez-Calvillo, S

    2015-11-01

    RNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

  18. 5-HT(1A) receptors transactivate the platelet-derived growth factor receptor type beta in neuronal cells.

    PubMed

    Kruk, Jeff S; Vasefi, Maryam S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2013-01-01

    In the absence of ligand, certain growth factor receptors can be activated via G-protein coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate platelet-derived growth factor (PDGF) β receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here we show that 5-HT can transiently increase the phosphorylation of PDGFβ receptors through 5-HT(1A) receptors in a time- and dose-dependent manner in SH-SY5Y neuroblastoma cells. 5-HT also transactivates PDGFβ receptors in primary cortical neurons. This transactivation pathway is pertussis-toxin sensitive and Src tyrosine kinase-dependent. This pathway is also dependent on phospholipase C activity and intracellular calcium signaling. Several studies involving PDGFβ receptor transactivation by GPCRs have also demonstrated a PDGFβ receptor-dependent increase in the phosphorylation of ERK1/2. Yet in SH-SY5Y cells, 5-HT treatment causes a PDGFβ receptor-independent increase in ERK1/2 phosphorylation. This crosstalk between 5-HT and PDGFβ receptors identifies a potentially important signaling link between the serotonergic system and growth factor signaling in neurons. PMID:23006663

  19. 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

  20. 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

  1. Proprotein Convertase Subtilisin/Kexin Type 7 (PCSK7) Is Essential for the Zebrafish Development and Bioavailability of Transforming Growth Factor β1a (TGFβ1a)*

    PubMed Central

    Turpeinen, Hannu; Oksanen, Anna; Kivinen, Virpi; Kukkurainen, Sampo; Uusimäki, Annemari; Rämet, Mika; Parikka, Mataleena; Hytönen, Vesa P.; Nykter, Matti; Pesu, Marko

    2013-01-01

    Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFβ1a. Consequently, tgfβ1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development. PMID:24178295

  2. 40 CFR Table W - 1A of Subpart W-Default Whole Gas Emission Factors for Onshore Petroleum and Natural Gas Production

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Emission Factors for Onshore Petroleum and Natural Gas Production W Table W Protection of Environment... Petroleum and Natural Gas Systems Definitions. Pt. 98, Subpt. W, Table W-1A Table W-1A of Subpart W—Default Whole Gas Emission Factors for Onshore Petroleum and Natural Gas Production Onshore petroleum...

  3. 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.

  4. An octopine synthase enhancer element directs tissue-specific expression and binds ASF-1, a factor from tobacco nuclear extracts.

    PubMed Central

    Fromm, H; Katagiri, F; Chua, N H

    1989-01-01

    We have investigated the expression pattern conferred by a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene in transgenic tobacco plants. Analysis of beta-glucuronidase expression driven by the ocs regulatory element revealed a pattern that is tissue-specific and developmentally regulated. In young seedlings, expression is confined primarily to root tips. In older seedlings, expression is stronger and becomes apparent also in the shoot apex. Insertion of a 16-base pair palindromic sequence (-193 to -178), which is included in the regulatory element, into an rbcS promoter results in the expression of rbcS in roots. The 16-base pair palindrome binds activation sequence factor (ASF)-1, a factor from tobacco nuclear extracts that interacts with the sequence between -83 to -63, designated as activation sequence (as)-1, of the cauliflower mosaic virus 35S promoter [Lam et al. (1989). Proc. Natl. Acad. Sci. USA 86, in press]. The in vivo expression patterns and in vitro binding properties of the ocs palindromic sequence are remarkably similar to those of the as-1 element of the cauliflower mosaic virus 35S promoter. These results suggest the involvement of ASF-1 in the transcriptional regulation of the ocs promoter and the 35S promoter. PMID:2562557

  5. Resistance to amoxicillin-clavulanate and its relation to virulence-related factors in Yersinia enterocolitica biovar 1A.

    PubMed

    Singhal, N; Kumar, M; Virdi, J S

    2016-01-01

    Recent studies have reported that the virulence factors (VFs) were detected more frequently in amoxicillin-clavulanate (AMC) susceptible clinical isolates of Escherichia coli. Here, we have evaluated the relationship between VFs and AMC-resistance phenotype in clinical isolates of Y. enterocolitica biovar 1A. The presence/absence of VFs was compared with their minimum inhibitory concentrations for AMC in strains of two serovars. We observed that the strains of the serovar O: 6, 30-6, 31 showed a similar relationship between the number of VFs and resistance to clavulanic acid as in E. coli but not of serovar O: 6, 30. Variations in the promoters/complete coding sequences (CCDSs) of β-lactamase gene (bla A) or the serological characteristics could not account for unusual susceptibility to AMC displayed by the strains of the serovar O: 6, 30. Therefore, we speculate that since the clinical strains of serovar O: 6, 30-6, 31 originated from the environment they were less exposed to antibiotics compared to clinical strains of serovar O: 6, 30. Thus, AMC susceptibility seems to be influenced by factors other than serotypes or promoters/CCDS of β-lactamase genes.

  6. Local Overexpression of V1a-Vasopressin Receptor Enhances Regeneration in Tumor Necrosis Factor-Induced Muscle Atrophy

    PubMed Central

    Costa, Alessandra; Toschi, Angelica; Murfuni, Ivana; Pelosi, Laura; Sica, Gigliola; Adamo, Sergio; Scicchitano, Bianca Maria

    2014-01-01

    Skeletal muscle atrophy occurs during disuse and aging, or as a consequence of chronic diseases such as cancer and diabetes. It is characterized by progressive loss of muscle tissue due to hypotrophic changes, degeneration, and an inability of the regeneration machinery to replace damaged myofibers. Tumor necrosis factor (TNF) is a proinflammatory cytokine known to mediate muscle atrophy in many chronic diseases and to inhibit skeletal muscle regeneration. In this study, we investigated the role of Arg-vasopressin-(AVP-)dependent pathways in muscles in which atrophy was induced by local overexpression of TNF. AVP is a potent myogenesis-promoting factor and is able to enhance skeletal muscle regeneration by stimulating Ca2+/calmodulin-dependent kinase and calcineurin signaling. We performed morphological and molecular analyses and demonstrated that local over-expression of the AVP receptor V1a enhances regeneration of atrophic muscle. By upregulating the regeneration/differentiation markers, modulating the inflammatory response, and attenuating fibrogenesis, the stimulation of AVP-dependent pathways creates a favourable environment for efficient and sustained muscle regeneration and repair even in the presence of elevated levels of TNF. This study highlights a novel in vivo role for AVP-dependent pathways, which may represent an interesting strategy to counteract muscle decline in aging or in muscular pathologies. PMID:24971321

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. Demonstration of cytotoxicity against wasps by pierisin-1: a possible defense factor in the cabbage white butterfly.

    PubMed

    Takahashi-Nakaguchi, Azusa; Matsumoto, Yasuko; Yamamoto, Masafumi; Iwabuchi, Kikuo; Totsuka, Yukari; Sugimura, Takashi; Wakabayashi, Keiji

    2013-01-01

    The cabbage white butterfly, Pieris rapae, produces pierisin-1, a protein inducing apoptosis of mammalian cells. In the present study, the biological activity of pierisin-1 as a protective agent against parasitic wasps for P. rapae was examined. Pierisin-1 caused detrimental effects on eggs and larvae of non-habitual parasitoids for P. rapae, Glyptapanteles pallipes, Cotesia kariyai and Cotesia plutellae at 1-100 µg/ml, levels essentially equivalent to those found in P. rapae larvae. In contrast, eggs and larvae of the natural parasitoid of P. rapae, Cotesia glomerata proved resistant to the toxicity of pierisin-1 through inhibition of pierisin-1 penetration of the surface layer. The expression level of pierisin-1 mRNA in the larvae of P. rapae was increased by parasitization by C. plutellae, whereas it was decreased by C. glomerata. In addition, C. plutellae was associated with elevation of activated pierisin-1 in the hemolymph. From these observations, it is suggested that pierisin-1 could contribute as a defense factor against parasitization by some type of wasps in P. rapae.

  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. Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

    PubMed

    Cheng, Yu-Jie; Kim, Myoung-Duck; Deng, Xi-Ping; Kwak, Sang-Soo; Chen, Wei

    2013-12-01

    IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes.

  16. Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

    PubMed

    Cheng, Yu-Jie; Kim, Myoung-Duck; Deng, Xi-Ping; Kwak, Sang-Soo; Chen, Wei

    2013-12-01

    IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes. PMID:24378636

  17. 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.

  18. 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

  19. 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

  20. Ancestral Mutations Acquired in Refrex-1, a Restriction Factor against Feline Retroviruses, during its Cooption and Domestication

    PubMed Central

    Ito, Jumpei; Baba, Takuya; Kawasaki, Junna

    2015-01-01

    ABSTRACT Endogenous retroviruses (ERVs) are remnants of ancestral retroviral infections of germ cells. Retroviral endogenization is an adaptation process for the host genome, and ERVs are gradually attenuated or inactivated by mutation. However, some ERVs that have been “domesticated” by their hosts eventually gain physiological functions, such as placentation or viral resistance. We previously reported the discovery of Refrex-1, a soluble antiretroviral factor in domestic cats that specifically inhibits infection by feline leukemia virus subgroup D (FeLV-D), a chimeric virus of FeLV, and a feline ERV, ERV-DC. Refrex-1 is a truncated envelope protein (Env) encoded by both ERV-DC7 and ERV-DC16 proviral loci. Here, we reconstituted ancestral and functional Env from ERV-DC7 and ERV-DC16 envelope genes (env) by inducing reverse mutations. Unexpectedly, ERV-DC7 and ERV-DC16 full-length Env (ERV-DC7 fl and ERV-DC16 fl), reconstructed by removing stop codons, did not produce infectious viral particles. ERV-DC7 fl and ERV-DC16 fl were highly expressed in cells but were not cleaved into surface subunits (SU) and transmembrane subunits, nor were they incorporated into virions. G407R/N427I-A429T and Y431D substitutions within the SU C-terminal domain of ERV-DC7 fl and ERV-DC16 fl, respectively, caused these dysfunctions. The residues glycine 407 and tyrosine 431 are relatively conserved among infectious gammaretroviruses, and their substitution causes the same dysfunctions as the tested retroviruses. Our results reveal that specific mutations within the SU C-terminal domain suppressed Env cleavage and incorporation into virions and indicate that these mutations contributed to the domestication of Refrex-1 through multistep events that occurred in the postintegration period. IMPORTANCE Domestic cats are colonized with various exogenous retroviruses (exRVs), such as feline leukemia virus (FeLV), and their genomes contain numerous ERVs, some of which are replication

  1. 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.

  2. 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.

  3. 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

  4. The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation.

    PubMed

    Shang, Yingli; Coppo, Maddalena; He, Teng; Ning, Fei; Yu, Li; Kang, Lan; Zhang, Bin; Ju, Chanyang; Qiao, Yu; Zhao, Baohong; Gessler, Manfred; Rogatsky, Inez; Hu, Xiaoyu

    2016-08-01

    Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription-initiation steps, and evidence for post-initiation regulation of inflammatory gene expression remains scarce. We found that the transcriptional repressor Hes1 suppressed production of CXCL1, a chemokine that is crucial for recruiting neutrophils. Hes1 negatively regulated neutrophil recruitment in vivo in a manner that was dependent on macrophage-produced CXCL1, and it attenuated the severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 did not involve modification of transcription initiation. Instead, Hes1 inhibited signal-induced recruitment of the positive transcription-elongation complex P-TEFb and thereby prevented phosphorylation of RNA polymerase II at Ser2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses that exerts its suppressive function by regulating transcription elongation.

  5. Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus

    PubMed Central

    Yang, Xiangcai; Li, Jiejing; Zeng, Wanli; Li, Chaocui; Mao, Bingyu

    2016-01-01

    Elongator protein 3 (Elp3) is the enzymatic unit of the elongator protein complex, a histone acetyltransferase complex involved in transcriptional elongation. It has long been shown to play an important role in cell migration; however, the underlying mechanism is unknown. Here, we showed that Elp3 is expressed in pre-migratory and migrating neural crest cells in Xenopus embryos, and knockdown of Elp3 inhibited neural crest cell migration. Interestingly, Elp3 binds Snail1 through its zinc-finger domain and inhibits its ubiquitination by β-Trcp without interfering with the Snail1/Trcp interaction. We showed evidence that Elp3-mediated stabilization of Snail1 was likely involved in the activation of N-cadherin in neural crest cells to regulate their migratory ability. Our findings provide a new mechanism for the function of Elp3 in cell migration through stabilizing Snail1, a master regulator of cell motility. PMID:27189455

  6. Nuclear factor XIIIa staining (clone AC-1A1 mouse monoclonal) is a sensitive and specific marker to discriminate sebaceous proliferations from other cutaneous clear cell neoplasms.

    PubMed

    Uhlenhake, Elizabeth E; Clark, Lindsey N; Smoller, Bruce R; Shalin, Sara C; Gardner, Jerad M

    2016-08-01

    Sebaceous carcinoma is a rare but serious malignancy that may be difficult to diagnose when poorly differentiated. Other epithelial tumors with clear cell change may mimic sebaceous carcinoma. Few useful or specific immunohistochemical markers for sebaceous differentiation are available. Nuclear staining with factor XIIIa (clone AC-1A1) was recently found to be a highly sensitive marker of sebaceous differentiation. We evaluated nuclear factor XIIIa (AC-1A1) staining in sebaceous neoplasms vs. other cutaneous clear cell tumors. We stained 27 sebaceous proliferations: sebaceous hyperplasia (7), sebaceous adenoma (8), sebaceoma (5), sebaceous carcinoma (7). We also stained 67 tumors with clear cell change: basal cell carcinoma (8), squamous cell carcinoma (8), hidradenoma (7), desmoplastic trichilemmoma (2), trichilemmoma (10), trichilemmal carcinoma (3), clear cell acanthoma (9), atypical fibroxanthoma (1), syringoma (8), trichoepithelioma (1), metastatic renal cell carcinoma (2), and nevi with balloon cell change (8). Nuclear factor XIIIa (AC-1A1) staining was present in 100% of sebaceous proliferations; 96% displayed strong staining. Non-sebaceous clear cell tumors were negative or only weakly positive with factor XIIIa (AC-1A1) in 95.5%; only 4.5% showed strong staining. This suggests that strong nuclear factor XIIIa (AC-1A1) staining is a sensitive and specific marker of sebaceous neoplasms vs. other clear cell tumors.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family.

    PubMed Central

    Oettgen, P; Alani, R M; Barcinski, M A; Brown, L; Akbarali, Y; Boltax, J; Kunsch, C; Munger, K; Libermann, T A

    1997-01-01

    We report here the isolation of a novel, highly tissue-restricted member of the ets transcription factor/oncogene family, ESE-1 (for epithelium-specific Ets), which has features distinct from those of any other ets-related factor. ESE-1 contains two putative DNA binding domains: an ETS domain, which is unique in that the 5' half shows relatively weak homology to known ets factors, and an A/T hook domain, found in HMG proteins and various other nuclear factors. In contrast to any known ets factors, ESE-1 is expressed exclusively in epithelial cells. ESE-1 expression is induced during terminal differentiation of the epidermis and in a primary human keratinocyte differentiation system. The keratinocyte terminal differentiation marker gene, SPRR2A, is a putative target for ESE-1, since SPRR2A expression during keratinocyte differentiation correlates with induction of ESE-1 expression, and ESE-1 binds with high affinity to and transactivates the ets binding site in the SPRR2A promoter. ESE-1 also binds to and transactivates the enhancer of the Endo A gene, a potential target for ESE-1 in simple epithelia. Due to the important role that other ets factors play in cellular differentiation, ESE-1 is expected to be a critical regulator of epithelial cell differentiation. PMID:9234700

  12. 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.

  13. 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

  14. Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

    PubMed Central

    Lee, Bomi; Wu, Cheng-Ying; Lin, Yi-Wei; Park, Sung Wook; Wei, Li-Na

    2016-01-01

    All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity. PMID:27166374

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Potential Risk Factors for the Onset of Complex Regional Pain Syndrome Type 1: A Systematic Literature Review

    PubMed Central

    Shipton, Edward A.; Mulder, Roger T.

    2015-01-01

    Anaesthetists in the acute and chronic pain teams are often involved in treating Complex Regional Pain Syndromes. Current literature about the risk factors for the onset of Complex Regional Pain Syndrome Type 1 (CRPS 1) remains sparse. This syndrome has a low prevalence, a highly variable presentation, and no gold standard for diagnosis. In the research setting, the pathogenesis of the syndrome continues to be elusive. There is a growing body of literature that addresses efficacy of a wide range of interventions as well as the likely mechanisms that contribute to the onset of CRPS 1. The objective for this systematic search of the literature focuses on determining the potential risk factors for the onset of CRPS 1. Eligible articles were analysed, dated 1996 to April 2014, and potential risk factors for the onset of CRPS 1 were identified from 10 prospective and 6 retrospective studies. Potential risk factors for the onset of CRPS 1 were found to include being female, particularly postmenopausal female, ankle dislocation or intra-articular fracture, immobilisation, and a report of higher than usual levels of pain in the early phases of trauma. It is not possible to draw definite conclusions as this evidence is heterogeneous and of mixed quality, relevance, and weighting strength against bias and has not been confirmed across multiple trials or in homogenous studies. PMID:25688265

  20. The interaction between eukaryotic initiation factor 1A and eIF5 retains eIF1 within scanning preinitiation complexes.

    PubMed

    Luna, Rafael E; Arthanari, Haribabu; Hiraishi, Hiroyuki; Akabayov, Barak; Tang, Leiming; Cox, Christian; Markus, Michelle A; Luna, Lunet E; Ikeda, Yuka; Watanabe, Ryosuke; Bedoya, Edward; Yu, Cathy; Alikhan, Shums; Wagner, Gerhard; Asano, Katsura

    2013-12-31

    Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

  1. PcFKH1, a novel regulatory factor from the forkhead family, controls the biosynthesis of penicillin in Penicillium chrysogenum.

    PubMed

    Domínguez-Santos, Rebeca; García-Estrada, Carlos; Kosalková, Katarina; Prieto, Carlos; Santamarta, Irene; Martín, Juan-Francisco

    2015-08-01

    Penicillin biosynthesis in Penicillium chrysogenum (re-identified as Penicillium rubens) is a good example of a biological process subjected to complex global regulatory networks and serves as a model to study fungal secondary metabolism. The winged-helix family of transcription factors recently described, which includes the forkhead type of proteins, is a key type of regulatory proteins involved in this process. In yeasts and humans, forkhead transcription factors are involved in different processes (cell cycle regulation, cell death control, pre-mRNA processing and morphogenesis); one member of this family of proteins has been identified in the P. chrysogenum genome (Pc18g00430). In this work, we have characterized this novel transcription factor (named PcFKH1) by generating knock-down mutants and overexpression strains. Results clearly indicate that PcFKH1 positively controls antibiotic biosynthesis through the specific interaction with the promoter region of the penDE gene, thus regulating penDE mRNA levels. PcFKH1 also binds to the pcbC promoter, but with low affinity. In addition, it also controls other ancillary genes of the penicillin biosynthetic process, such as phlA (encoding phenylacetyl CoA ligase) and ppt (encoding phosphopantetheinyl transferase). PcFKH1 also plays a role in conidiation and spore pigmentation, but it does not seem to be involved in hyphal morphology or cell division in the improved laboratory reference strain Wisconsin 54-1255. A genome-wide analysis of processes putatively coregulated by PcFKH1 and PcRFX1 (another winged-helix transcription factor) in P. chrysogenum provided evidence of the global effect of these transcription factors in P. chrysogenum metabolism.

  2. SWI/SNF factors required for cellular resistance to DNA damage include ARID1A and ARID1B and show interdependent protein stability.

    PubMed

    Watanabe, Reiko; Ui, Ayako; Kanno, Shin-Ichiro; Ogiwara, Hideaki; Nagase, Takahiro; Kohno, Takashi; Yasui, Akira

    2014-05-01

    The SWI/SNF chromatin-remodeling family contains various protein complexes, which regulate gene expression during cellular development and influence DNA damage response in an ATP- and complex-dependent manner, of which details remain elusive. Recent human genome sequencing of various cancer cells revealed frequent mutations in SWI/SNF factors, especially ARID1A, a variant subunit in the BRG1-associated factor (BAF) complex of the SWI/SNF family. We combined live-cell analysis and gene-suppression experiments to show that suppression of either ARID1A or its paralog ARID1B led to reduced nonhomologous end joining activity of DNA double-strand breaks (DSB), decreased accumulation of KU70/KU80 proteins at DSB, and sensitivity to ionizing radiation, as well as to cisplatin and UV. Thus, in contrast to transcriptional regulation, both ARID1 proteins are required for cellular resistance to various types of DNA damage, including DSB. The suppression of other SWI/SNF factors, namely SNF5, BAF60a, BAF60c, BAF155, or BAF170, exhibits a similar phenotype. Of these factors, ARID1A, ARID1B, SNF5, and BAF60c are necessary for the immediate recruitment of the ATPase subunit of the SWI/SNF complex to DSB, arguing that both ARID1 proteins facilitate the damage response of the complex. Finally, we found interdependent protein stability among the SWI/SNF factors, suggesting their direct interaction within the complex and the reason why multiple factors are frequently lost in parallel in cancer cells. Taken together, we show that cancer cells lacking in the expression of certain SWI/SNF factors, including ARID1A, are deficient in DNA repair and potentially vulnerable to DNA damage.

  3. 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

  4. 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.

  5. 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...

  6. 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

  7. FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

    PubMed

    Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N

    1999-03-01

    The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.

  8. FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

    PubMed Central

    Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N

    1999-01-01

    The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors. PMID:9973611

  9. 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

  10. Typical and severe tumor necrosis factor receptor-associated periodic syndrome in the absence of mutations in the TNFRSF1A gene: a case series.

    PubMed

    Cantarini, Luca; Lucherini, Orso Maria; Cimaz, Rolando; Rigante, Donato; Baldari, Cosima Tatiana; Laghi Pasini, Franco; Galeazzi, Mauro

    2012-12-01

    Tumor necrosis factor receptor-1-associated periodic syndrome (TRAPS) is the most common autosomal dominant autoinflammatory disorder and is caused by mutations in the TNFRSF1A gene encoding the 55-kDa receptor for tumor necrosis factor (TNF)-α. TRAPS is characterized by recurrent attacks of fever, typically lasting from 1 to 3 weeks. In addition to fever, common clinical features include periorbital edema, a migratory erythematous plaque simulating erysipela with underlying myalgia, and arthralgia or arthritis. Serosal membrane inflammation is also a common feature, usually in the form of polyserositis. To date, at least 40 different TNFRSF1A mutations have been identified, but few patients with symptoms highly suggestive of TRAPS with no mutations in the TNFRSF1A gene have recently been described, thus suggesting that not all mutations are yet known or that alternative mechanisms might be involved in the pathogenesis of the disease. We report on three such patients here.

  11. 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

  12. 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.

  13. 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.

  14. Rac1 Controls the Subcellular Localization of the Rho Guanine Nucleotide Exchange Factor Net1A To Regulate Focal Adhesion Formation and Cell Spreading

    PubMed Central

    Carr, Heather S.; Morris, Christopher A.; Menon, Sarita; Song, Eun Hyeon

    2013-01-01

    RhoA is overexpressed in human cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA guanine nucleotide exchange factor that is overexpressed in human cancer. It encodes two isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1 proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1 isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1 isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from proteasome-mediated degradation. Replating cells on collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on collagen, myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1 isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion. PMID:23184663

  15. Molecular cloning and function analysis of insulin-like growth factor-binding protein 1a in blunt snout bream (Megalobrama amblycephala).

    PubMed

    Tian, Yu-Mei; Chen, Jie; Tao, Yang; Jiang, Xia-Yun; Zou, Shu-Ming

    2014-07-01

    Insulin-like growth factor-binding protein 1 (IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream (Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions. PMID:25017749

  16. EjODO1, a MYB Transcription Factor, Regulating Lignin Biosynthesis in Developing Loquat (Eriobotrya japonica) Fruit

    PubMed Central

    Zhang, Jing; Ge, Hang; Zang, Chen; Li, Xian; Grierson, Donald; Chen, Kun-song; Yin, Xue-ren

    2016-01-01

    Lignin is important for plant secondary cell wall formation and participates in resistance to various biotic and abiotic stresses. Loquat undergoes lignification not only in vegetative tissues but also in flesh of postharvest fruit, which adversely affects consumer acceptance. Thus, researches on lignin biosynthesis and regulation are important to understand loquat fruit lignification. In loquat, a gene encoding an enzyme in the lignin biosynthesis pathway, Ej4CL1, was reported to be regulated by transcription factors, including EjMYB1, EjMYB2, EjMYB8, and EjAP2-1, knowledge of this process is still limited. With the aim of identifying novel transcriptional factors controlling lignin biosynthesis in loquat, the promoter of Ej4CL1 was utilized to screen a cDNA library by yeast one hybrid assay. A novel R2R3 MYB, named EjODO1, was identified. Real-time PCR analyses indicated that EjODO1 is highly expressed in lignified stems and roots. During fruit development, expression of EjODO1 decreased along with the reduction of lignin content and became undetectable in mature ripe fruit. Thus, EjODO1 is likely to be involved in lignification of vegetative organs and early fruit development but not in mature fruit or postharvest lignification. Dual-luciferase assay indicated that EjODO1 could trans-activate promoters of lignin biosynthesis genes, such as EjPAL1, Ej4CL1, and Ej4CL5 and transient overexpression of EjODO1 triggered lignin biosynthesis. These results indicate a role for EjODO1 in regulating lignin biosynthesis in loquat which is different from the previously characterized transcription factors. PMID:27695460

  17. EjODO1, a MYB Transcription Factor, Regulating Lignin Biosynthesis in Developing Loquat (Eriobotrya japonica) Fruit

    PubMed Central

    Zhang, Jing; Ge, Hang; Zang, Chen; Li, Xian; Grierson, Donald; Chen, Kun-song; Yin, Xue-ren

    2016-01-01

    Lignin is important for plant secondary cell wall formation and participates in resistance to various biotic and abiotic stresses. Loquat undergoes lignification not only in vegetative tissues but also in flesh of postharvest fruit, which adversely affects consumer acceptance. Thus, researches on lignin biosynthesis and regulation are important to understand loquat fruit lignification. In loquat, a gene encoding an enzyme in the lignin biosynthesis pathway, Ej4CL1, was reported to be regulated by transcription factors, including EjMYB1, EjMYB2, EjMYB8, and EjAP2-1, knowledge of this process is still limited. With the aim of identifying novel transcriptional factors controlling lignin biosynthesis in loquat, the promoter of Ej4CL1 was utilized to screen a cDNA library by yeast one hybrid assay. A novel R2R3 MYB, named EjODO1, was identified. Real-time PCR analyses indicated that EjODO1 is highly expressed in lignified stems and roots. During fruit development, expression of EjODO1 decreased along with the reduction of lignin content and became undetectable in mature ripe fruit. Thus, EjODO1 is likely to be involved in lignification of vegetative organs and early fruit development but not in mature fruit or postharvest lignification. Dual-luciferase assay indicated that EjODO1 could trans-activate promoters of lignin biosynthesis genes, such as EjPAL1, Ej4CL1, and Ej4CL5 and transient overexpression of EjODO1 triggered lignin biosynthesis. These results indicate a role for EjODO1 in regulating lignin biosynthesis in loquat which is different from the previously characterized transcription factors.

  18. The effect of physicochemical factors on the self-association of HMGB1: A surface plasmon resonance study.

    PubMed

    Anggayasti, Wresti L; Mancera, Ricardo L; Bottomley, Steven; Helmerhorst, Erik

    2016-11-01

    HMGB1 triggers proinflammatory reactions by interacting extracellularly with various receptors. HMGB1 also acts in the nucleus by interacting with DNA and controlling DNA transcription, a process which involves its self-association. The self-association of HMGB1 was characterized using surface plasmon resonance (SPR). A dimer/tetramer binding model was developed that provided a good fit to the SPR sensorgrams and enabled the kinetics of self-association of different HMGB1 oligomers to be evaluated under a variety of physicochemical conditions. The formation of HMGB1 tetramers, and not dimers, was strongly influenced by ionic strength. HMGB1 self-association increased as the pH was decreased from 7.4 to 4.8 but was abolished at pH4.0, suggesting the involvement of acidic amino acids of HMGB1 in its self-association. HMGB1 dimers were found to predominate in the absence of zinc, but addition of zinc promoted the formation of HMGB1 tetramers. More reducing conditions favored dimerization but diminished tetramer formation. In contrast, oxidizing conditions favored tetramer formation. Physicochemical factors modulate the extent of self-association of HMGB1. We speculate that HMGB1 dimers may preferentially bind DNA, whereas HMGB1 tetramers may promote inflammatory responses by binding to RAGE and TLRs. The self-association of HMGB1, regulated by variations of physicochemical factors, may influence its roles in DNA rearrangement and regulation of pathophysiological diseases. PMID:27476953

  19. The effect of physicochemical factors on the self-association of HMGB1: A surface plasmon resonance study.

    PubMed

    Anggayasti, Wresti L; Mancera, Ricardo L; Bottomley, Steven; Helmerhorst, Erik

    2016-11-01

    HMGB1 triggers proinflammatory reactions by interacting extracellularly with various receptors. HMGB1 also acts in the nucleus by interacting with DNA and controlling DNA transcription, a process which involves its self-association. The self-association of HMGB1 was characterized using surface plasmon resonance (SPR). A dimer/tetramer binding model was developed that provided a good fit to the SPR sensorgrams and enabled the kinetics of self-association of different HMGB1 oligomers to be evaluated under a variety of physicochemical conditions. The formation of HMGB1 tetramers, and not dimers, was strongly influenced by ionic strength. HMGB1 self-association increased as the pH was decreased from 7.4 to 4.8 but was abolished at pH4.0, suggesting the involvement of acidic amino acids of HMGB1 in its self-association. HMGB1 dimers were found to predominate in the absence of zinc, but addition of zinc promoted the formation of HMGB1 tetramers. More reducing conditions favored dimerization but diminished tetramer formation. In contrast, oxidizing conditions favored tetramer formation. Physicochemical factors modulate the extent of self-association of HMGB1. We speculate that HMGB1 dimers may preferentially bind DNA, whereas HMGB1 tetramers may promote inflammatory responses by binding to RAGE and TLRs. The self-association of HMGB1, regulated by variations of physicochemical factors, may influence its roles in DNA rearrangement and regulation of pathophysiological diseases.

  20. NIK1, a host factor specialized in antiviral defense or a novel general regulator of plant immunity?

    PubMed

    Machado, Joao P B; Brustolini, Otavio J B; Mendes, Giselle C; Santos, Anésia A; Fontes, Elizabeth P B

    2015-11-01

    NIK1 is a receptor-like kinase involved in plant antiviral immunity. Although NIK1 is structurally similar to the plant immune factor BAK1, which is a key regulator in plant immunity to bacterial pathogens, the NIK1-mediated defenses do not resemble BAK1 signaling cascades. The underlying mechanism for NIK1 antiviral immunity has recently been uncovered. NIK1 activation mediates the translocation of RPL10 to the nucleus, where it interacts with LIMYB to fully down-regulate translational machinery genes, resulting in translation inhibition of host and viral mRNAs and enhanced tolerance to begomovirus. Therefore, the NIK1 antiviral immunity response culminates in global translation suppression, which represents a new paradigm for plant antiviral defenses. Interestingly, transcriptomic analyses in nik1 mutant suggest that NIK1 may suppress antibacterial immune responses, indicating a possible opposite effect of NIK1 in bacterial and viral infections.

  1. 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.

  2. [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.

  3. 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.

  4. Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish

    PubMed Central

    Cheng, Christina N.; Wingert, Rebecca A.

    2014-01-01

    The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study this renal development process because its nephrons contain segments akin to other vertebrates, including the proximal convoluted and straight tubules (PCT, PST). The zebrafish pronephros is also associated with the corpuscles of Stannius (CS), endocrine glands that regulate calcium and phosphate homeostasis, but whose ontogeny from renal progenitors is largely mysterious. Initial patterning of zebrafish renal progenitors in the intermediate mesoderm (IM) involves the formation of rostral and caudal domains, the former being reliant on retinoic acid (RA) signaling, and the latter being repressed by elevated RA levels. Here, using expression profiling to gain new insights into nephrogenesis, we discovered that the gene single minded family bHLH transcription factor 1a (sim1a) is dynamically expressed in the renal progenitors—first marking the caudal domain, then becoming restricted to the proximal segments, and finally exhibiting specific CS expression. In loss of function studies, sim1a knockdown expanded the PCT and abrogated both the PST and CS populations. Conversely, overexpression of sim1a modestly expanded the PST and CS, while it reduced the PCT. These results show that sim1a activity is necessary and partially sufficient to induce PST and CS fates, and suggest that sim1a may inhibit PCT fate and/or negotiate the PCT/PST boundary. Interestingly, the sim1a expression domain in renal progenitors is responsive to altered levels of RA, suggesting that RA regulates sim1a, directly or indirectly, during nephrogenesis. sim1a deficient embryos treated with exogenous RA formed nephrons that were predominantly composed of PCT segments, but lacked the enlarged PST observed in RA treated wild-types, indicating that RA is not sufficient to rescue the PST in the absence of sim1a expression. Alternately

  5. Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish.

    PubMed

    Cheng, Christina N; Wingert, Rebecca A

    2015-03-01

    The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study this renal development process because its nephrons contain segments akin to other vertebrates, including the proximal convoluted and straight tubules (PCT, PST). The zebrafish pronephros is also associated with the corpuscles of Stannius (CS), endocrine glands that regulate calcium and phosphate homeostasis, but whose ontogeny from renal progenitors is largely mysterious. Initial patterning of zebrafish renal progenitors in the intermediate mesoderm (IM) involves the formation of rostral and caudal domains, the former being reliant on retinoic acid (RA) signaling, and the latter being repressed by elevated RA levels. Here, using expression profiling to gain new insights into nephrogenesis, we discovered that the gene single minded family bHLH transcription factor 1a (sim1a) is dynamically expressed in the renal progenitors-first marking the caudal domain, then becoming restricted to the proximal segments, and finally exhibiting specific CS expression. In loss of function studies, sim1a knockdown expanded the PCT and abrogated both the PST and CS populations. Conversely, overexpression of sim1a modestly expanded the PST and CS, while it reduced the PCT. These results show that sim1a activity is necessary and partially sufficient to induce PST and CS fates, and suggest that sim1a may inhibit PCT fate and/or negotiate the PCT/PST boundary. Interestingly, the sim1a expression domain in renal progenitors is responsive to altered levels of RA, suggesting that RA regulates sim1a, directly or indirectly, during nephrogenesis. sim1a deficient embryos treated with exogenous RA formed nephrons that were predominantly composed of PCT segments, but lacked the enlarged PST observed in RA treated wild-types, indicating that RA is not sufficient to rescue the PST in the absence of sim1a expression. Alternately

  6. Loss of CEACAM1, a Tumor-Associated Factor, Attenuates Post-infarction Cardiac Remodeling by Inhibiting Apoptosis

    PubMed Central

    Wang, Yan; Chen, Yanmei; Yan, Yi; Li, Xinzhong; Chen, Guojun; He, Nvqin; Shen, Shuxin; Chen, Gangbin; Zhang, Chuanxi; Liao, Wangjun; Liao, Yulin; Bin, Jianping

    2016-01-01

    Carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) is a tumor-associated factor that is known to be involved in apoptosis, but the role of CEACAM1 in cardiovascular disease is unclear. We aims to investigate whether CEACAM1 influences cardiac remodeling in mice with myocardial infarction (MI) and hypoxia-induced cardiomyocyte injury. Both serum in patients and myocardial CEACAM1 levels in mice were significantly increased in response to MI, while levels were elevated in neonatal rat cardiomyocytes (NRCs) exposed to hypoxia. Eight weeks after MI, a lower mortality rate, improved cardiac function, and less cardiac remodeling in CEACAM1 knock-out (KO) mice than in their wild-type (WT) littermates were observed. Moreover, myocardial expression of mitochondrial Bax, cytosolic cytochrome C, and cleaved caspase-3 was significantly lower in CEACAM1 KO mice than in WT mice. In cultured NRCs exposed to hypoxia, recombinant human CEACAM1 (rhCEACAM1) reduced mitochondrial membrane potential, upregulated mitochondrial Bax, increased cytosolic cytochrome C and cleaved caspase-3, and consequently increased apoptosis. RhCEACAM1 also increased the levels of GRP78 and CHOP in NRCs with hypoxia. All of these effects were abolished by silencing CEACAM1. Our study indicates that CEACAM1 exacerbates hypoxic cardiomyocyte injury and post-infarction cardiac remodeling by enhancing cardiomyocyte mitochondrial dysfunction and endoplasmic reticulum stress-induced apoptosis. PMID:26911181

  7. Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes.

    PubMed

    Massari, M E; Rivera, R R; Voland, J R; Quong, M W; Breit, T M; van Dongen, J J; de Smit, O; Murre, C

    1998-06-01

    Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination. Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library. Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis. Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells. ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A. Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines. ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells. ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.

  8. OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress.

    PubMed

    Wehner, Karen A; Schütz, Sylvia; Sarnow, Peter

    2010-04-01

    Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

  9. Ccpg1, a Novel Scaffold Protein That Regulates the Activity of the Rho Guanine Nucleotide Exchange Factor Dbs▿

    PubMed Central

    Kostenko, Elena V.; Olabisi, Oyenike O.; Sahay, Sutapa; Rodriguez, Pedro L.; Whitehead, Ian P.

    2006-01-01

    Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member. PMID:17000758

  10. Ser¹¹⁹ phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor.

    PubMed

    Soares-dos-Reis, Ricardo; Pessoa, Ana S; Matos, Mariana R; Falcão, Miguel; Mendes, Vera M; Manadas, Bruno; Monteiro, Filipe A; Lima, Deolinda; Reguenga, Carlos

    2014-05-01

    PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹⁹ and Ser²³⁸. Phosphorylation at Ser¹¹⁹ is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹⁹ phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.

  11. FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

    PubMed Central

    Lorenzi, M V; Horii, Y; Yamanaka, R; Sakaguchi, K; Miki, T

    1996-01-01

    A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 PMID:8799135

  12. TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco.

    PubMed

    Hu, Wei; Huang, Chao; Deng, Xiaomin; Zhou, Shiyi; Chen, Lihong; Li, Yin; Wang, Cheng; Ma, Zhanbing; Yuan, Qianqian; Wang, Yan; Cai, Rui; Liang, Xiaoyu; Yang, Guangxiao; He, Guangyuan

    2013-08-01

    Abscisic acid (ABA)-, stress-, and ripening-induced (ASR) proteins are reported to be involved in abiotic stresses. However, it is not known whether ASR genes confer drought stress tolerance by utilizing the antioxidant system. In this study, a wheat ASR gene, TaASR1, was cloned and characterized. TaASR1 transcripts increased after treatments with PEG6000, ABA and H(2)O(2). Overexpression of TaASR1 in tobacco resulted in increased drought/osmotic tolerance, which was demonstrated that transgenic lines had lesser malondialdehyde (MDA), ion leakage (IL) and reactive oxygen species (ROS), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities than wild type (WT) under drought stress. Overexpression of TaASR1 in tobacco also enhanced the expression of ROS-related and stress-responsive genes under osmotic stress. In addition, transgenic lines exhibited improved tolerance to oxidative stress by retaining more effective antioxidant system. Finally, TaASR1 was localized in the cell nucleus and functioned as a transcriptional activator. Taken together, our results showed that TaASR1 functions as a positive factor under drought/osmotic stress, involved in the regulation of ROS homeostasis by activating antioxidant system and transcription of stress-associated genes. PMID:23356734

  13. 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

  14. 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

  15. Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A.

    PubMed

    Xu, Yan-Hua; Wang, Jia-Wei; Wang, Shui; Wang, Jian-Ying; Chen, Xiao-Ya

    2004-05-01

    The cotton (+)-delta-cadinene synthase (CAD1), a sesquiterpene cyclase, catalyzes a branch-point step leading to biosynthesis of sesquiterpene phytoalexins, including gossypol. CAD1-A is a member of CAD1 gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated several WRKY cDNAs from Gossypium arboreum. One of them, GaWRKY1, encodes a protein containing a single WRKY domain and a putative N-terminal Leu zipper. Similar to genes encoding enzymes of cotton sesquiterpene pathway, GaWRKY1 was down-regulated in a glandless cotton cultivar that contained much less gossypol. GaWRKY1 showed a temporal and spatial pattern of expression comparable to that of CAD1-A in various aerial organs examined, including sepal, stigma, anther, and developing seeds. In suspension cells, expression of both GaWRKY1 and CAD1-A genes and biosynthesis of sesquiterpene aldehydes were strongly induced by a fungal elicitor preparation and methyl jasmonate. GaWRKY1 interacted with the 3x W-box derived from CAD1-A promoter in yeast (Saccharomyces cerevisiae) one-hybrid system and in vitro. Furthermore, in transgenic Arabidopsis plants, overexpression of GaWRKY1 highly activated the CAD1-A promoter, and transient assay in tobacco (Nicotiana tabacum) leaves demonstrated that W-box was required for this activation. These results suggest that GaWRKY1 participates in regulation of sesquiterpene biosynthesis in cotton, and CAD1-A is a target gene of this transcription factor. PMID:15133151

  16. Insulin-like growth factor 1: A novel treatment for the protection or regeneration of cochlear hair cells.

    PubMed

    Yamahara, Kohei; Yamamoto, Norio; Nakagawa, Takayuki; Ito, Juichi

    2015-12-01

    Sensorineural hearing loss (SNHL) is mainly caused by cochlear hair cell damage. Because cochlear hair cells and supporting cells lose their ability to proliferate in postnatal mammals, SNHL was thought to be an intractable disease. The maintenance of hair cell and supporting cell numbers after cochlear injury is therefore important for the treatment of sensorineural hearing loss. To achieve such treatment, protection and/or regeneration of hair cells is necessary. Progress in cochlear injury research, developmental biology, and regenerative medicine has led to the discovery of cochlear hair cells being protected or regenerated not only by direct reaction of hair cells themselves but also by that of supporting cells. Insulin-like growth factor 1 (IGF1) is considered a novel and potent treatment for SNHL based on the findings of various in vivo and in vitro experiments and clinical trials. The application of IGF1 maintains hair cell number of postnatal mammalian cochleae after various kinds of ototoxicity including aminoglycoside treatment, noise exposure, and ischemia. The positive effects of IGF1 on hair cell damage have been confirmed with in vivo animal experiments; hearing recovery in patients with sudden sensorineural hearing loss refractory to systemic glucocorticoid treatment has also been shown to occur following IGF1 treatment. The mechanisms of IGF1-induced maintenance of hair cell number have been investigated using a cochlear explant culture system, which demonstrated that IGF1 acts on supporting cells, leading to the inhibition of hair cell apoptosis and the proliferation of supporting cells. Netrin1 has furthermore been identified as one of the effectors whose expression is increased by IGF1 treatment.

  17. 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.

  18. 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

  19. 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

  20. Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity.

    PubMed

    Yang, Kun-Lin; Chang, Wen-Teng; Hung, Kuo-Chen; Li, Eric I C; Chuang, Chia-Chang

    2008-08-22

    Transforming growth factor-beta1 (TGF-beta1) mediates expression of collagen 1A2 (Col 1A2) gene via a synergistic cooperation between Smad2/Smad3 and Sp1, both act on the Col 1A2 gene promoter. In our previous study, we reported that a retinoic acid derivative obtained from Phellinus linteus (designated PL) antagonizes TGF-beta-induced liver fibrosis through regulation of ROS and calcium influx. In this continuing study we seek further the effect of PL on the Smad signaling pathway. We used a Col 1A2 promoter-luciferase construct to study the action of PL on Smad through TGF-beta. We found that PL decreases the promoter activity of Col 1A2, hinders the translocalization of phosphorylated Smad2/3-Smad 4 complex from cytosol into nucleus and inhibits Sp1 binding activity. These results suggest that PL inhibits TGF-beta1-induced Col 1A2 promoter activity through blocking ROS and calcium influx as well as impeding Sp1 binding and translocalization of pSmad 2/3-Smad4 complex into nucleus.

  1. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

    SciTech Connect

    Kim, Hyung Gyun; Han, Eun Hee; Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo; Jeong, Hye Gwang

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

  2. The ATPase Inhibitory Factor 1 (IF1): A master regulator of energy metabolism and of cell survival.

    PubMed

    García-Bermúdez, Javier; Cuezva, José M

    2016-08-01

    In this contribution we summarize most of the findings reported for the molecular and cellular biology of the physiological inhibitor of the mitochondrial H(+)-ATP synthase, the engine of oxidative phosphorylation (OXPHOS) and gate of cell death. We first describe the structure and major mechanisms and molecules that regulate the activity of the ATP synthase placing the ATPase Inhibitory Factor 1 (IF1) as a major determinant in the regulation of the activity of the ATP synthase and hence of OXPHOS. Next, we summarize the post-transcriptional mechanisms that regulate the expression of IF1 and emphasize, in addition to the regulation afforded by the protonation state of histidine residues, that the activity of IF1 as an inhibitor of the ATP synthase is also regulated by phosphorylation of a serine residue. Phosphorylation of S39 in IF1 by the action of a mitochondrial cAMP-dependent protein kinase A hampers its interaction with the ATP synthase, i.e., only dephosphorylated IF1 interacts with the enzyme. Upon IF1 interaction with the ATP synthase both the synthetic and hydrolytic activities of the engine of OXPHOS are inhibited. These findings are further placed into the physiological context to stress the emerging roles played by IF1 in metabolic reprogramming in cancer, in hypoxia and in cellular differentiation. We review also the implication of IF1 in other cellular situations that involve the malfunctioning of mitochondria. Special emphasis is given to the role of IF1 as driver of the generation of a reactive oxygen species signal that, emanating from mitochondria, is able to reprogram the nucleus of the cell to confer by various signaling pathways a cell-death resistant phenotype against oxidative stress. Overall, our intention is to highlight the urgent need of further investigations in the molecular and cellular biology of IF1 and of its target, the ATP synthase, to unveil new therapeutic strategies in human pathology. This article is part of a Special Issue

  3. trans-dominant mutants of E1A provide genetic evidence that the zinc finger of the trans-activating domain binds a transcription factor.

    PubMed Central

    Webster, L C; Ricciardi, R P

    1991-01-01

    The 289R E1A protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by E1A is mediated through cellular transcription factors. In particular, the ability of the trans-dominant E1A point mutant hr5 (Ser-185 to Asn) to inhibit wild-type E1A trans-activation was proposed to result from the sequestration of a cellular factor. Using site-directed mutagenesis, we individually replaced every residue within and flanking the trans-activating domain with a conservative amino acid, revealing 16 critical residues. Six of the individual substitutions lying in a contiguous stretch C terminal to the zinc finger (carboxyl region183-188) imparted a trans-dominant phenotype. trans-Dominance was even produced by deletion of the entire carboxyl region183-188. Conversely, an intact finger region147-177 was absolutely required for trans-dominance, since second-site substitution of every critical residue in this region abrogated the trans-dominant phenotype of the hr5 protein. These data indicate that the finger region147-177 bind a limiting cellular transcription factor and that the carboxyl region183-188 provides a separate and essential function. In addition, we show that four negatively charged residues within the trans-activating domain do not comprise a distinct acidic activating region. We present a model in which the trans-activating domain of E1A binds to two different cellular protein targets through the finger and carboxyl regions. Images PMID:1831535

  4. Methylation at a transcription factor-binding site on the 5-HT1A receptor gene correlates with negative symptom treatment response in first episode schizophrenia.

    PubMed

    Tang, Hao; Dalton, Caroline F; Srisawat, Umarat; Zhang, Zhi Jun; Reynolds, Gavin P

    2014-04-01

    Individual variability and inadequate response of negative symptoms are major limitations of antipsychotic treatment in schizophrenia. A functional polymorphism, rs6295, in the 5-HT1A-receptor gene (HTR1A) contributes to this variability in negative symptom response. The DNA sequence containing rs6295 is rich in cytosine methylation (CpG) sites; CpG methylation is an epigenetic factor that, like rs6295, can modify transcriptional control. To investigate whether DNA methylation influences response to antipsychotic treatment, we determined methylation at CpG sites close to rs6295 in DNA from 82 Chinese subjects with a first psychotic episode. Methylation of one CpG site within a recognition sequence for HES transcriptional repressors was found to correlate with changes in total PANSS score (p = 0.006) and negative factor sub-score (p < 0.001) following 10 wk initial antipsychotic treatment, as well as with baseline negative factor score (p = 0.019); the effect on symptom change remained after correction for this baseline score. An effect of rs6295 on negative symptom response was not seen in this sample, which may not have provided sufficient power for the pharmacogenetic association. These preliminary results indicate that epigenetic modification of transcriptional regulation by specific cytosine methylation may modulate HTR1A expression, resulting in effects on emotional dysfunction and negative symptom response to antipsychotic treatment. PMID:24331356

  5. Mapping of the gene for the p60 subunit of the human chromatin assembly factor (CAF1A) to the Down syndrome region of chromosome 21

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.; Antonarakis, S.E.

    1996-04-15

    Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescence in situ hybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakov et al. YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome. 22 refs., 1 fig.

  6. 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

  7. 17β-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y cells.

    PubMed

    Adeosun, Samuel O; Albert, Paul R; Austin, Mark C; Iyo, Abiye H

    2012-05-01

    Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17βE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17βE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17βE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17βE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17βE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

  8. 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.

  9. 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.

  10. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-09-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. PMID:2143540

  11. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed Central

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-01-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. Images PMID:2143540

  12. 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

  13. Truncation of the A,A(∗),A' helices segment impairs the actin bundling activity of mammalian eEF1A1.

    PubMed

    Vlasenko, Dmytro O; Novosylna, Oleksandra V; Negrutskii, Boris S; El'skaya, Anna V

    2015-05-01

    Translation elongation factor eEF1A is a G-protein which has a crucial role in the ribosomal polypeptide elongation and possesses a number of non-translational functions. Here, we show that the A,A(∗),A' helices segment of mammalian eEF1A is dispensable for the eEF1A*eEF1Bα complex formation. The A,A(∗),A' helices region did not interact with actin; however, its removal eliminates the actin bundling activity of eEF1A, probably due to the destruction of a dimeric structure of eEF1A. The translation function of monomers and the actin-bundling function of dimers of mammalian eEF1A is suggested.

  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. Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing.

    PubMed

    Himmelspach, M; Cavaloc, Y; Chebli, K; Stévenin, J; Gattoni, R

    1995-10-01

    Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.

  16. Arabidopsis heat shock factor HsfA1a directly senses heat stress, pH changes, and hydrogen peroxide via the engagement of redox state.

    PubMed

    Liu, Yanfang; Zhang, Cuixian; Chen, Juan; Guo, Lihong; Li, Xiaolu; Li, Wenpeng; Yu, Zefen; Deng, Jingshi; Zhang, Pengyuan; Zhang, Keqin; Zhang, Lemin

    2013-03-01

    Arabidopsis heat shock factor HsfA1a is present in a latent, monomeric state under normal conditions; its activation involves heat stress-induced trimerization, binding to heat shock element in target promoters, and the acquisition of transcriptional competence. HsfA1a is an important regulator for heat stress-induced gene expression and thermotolerance. However, it is not clear whether HsfA1a is directly activated by stress and the mechanisms of the stress signaling are poorly understood. We analyzed HsfA1a activation by trimerization and DNA-binding assays in vitro and in vivo in response to heat stress, low/high pH, and hydrogen peroxide treatments. Our results show that purified recombinant HsfA1a was activated by these stress treatments in vitro. The same treatments also induced the binding to HSP18.2 and HSP70 promoters as examined by chromatin immunoprecipitation, and the HsfA1a DNA binding paralleled the mRNA expression of its target genes induced by different stresses. Stress-induced DNA-binding could be reversed, both in vitro and in vivo, by subsequent incubation with reducing agents (DTT, NADPH). These data suggest that HsfA1a can directly sense stress and become activated, and this process is dependent on the redox state. An N-terminal deletion of the amino acid residues from 48 to 74 negatively affected pH- and hydrogen peroxide-, but not heat-stress sensing.

  17. Glutamate regulates eEF1A phosphorylation and ribosomal transit time in Bergmann glial cells.

    PubMed

    Barrera, Iliana; Flores-Méndez, Marco; Hernández-Kelly, Luisa C; Cid, Luis; Huerta, Miriam; Zinker, Samuel; López-Bayghen, Esther; Aguilera, José; Ortega, Arturo

    2010-12-01

    Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes. However, much less is known about the molecular events that modify the translational machinery upon exposure to this neurotransmitter. In a glial model of cerebellar cultured Bergmann cells, glutamate induces a biphasic effect on [(35)S]-methionine incorporation into proteins that suggests that the elongation phase of protein biosynthesis is the target for regulation. Indeed, after a 15 min exposure to glutamate a transient increase in elongation factor 2 phosphorylation has been reported, an effect mediated through the activation of the elongation factor 2 kinase. In this contribution, we sought to characterize the phosphorylation status of the eukaryotic elongation factor 1A (eEF1A) and the ribosomal transit time under glutamate exposure. A dose-dependent increase in eEF1A phosphorylation was found after a 60 min glutamate treatment; this phenomenon is Ca(2+)/CaM dependent, blocked with Src and phosphatidyl-inositol 3-kinase inhibitors and with rapamicyn. Concomitantly, the ribosomal transit time was increased with a 15 min glutamate exposure. After 60 more minutes, the average time used by the ribosomes to complete a polypeptide chain had almost returned to its initial level. These results strongly suggest that glutamate exerts an exquisite time-dependent translational control in glial cells, a process that might be critical for glia-neuron interactions.

  18. 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

  19. 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.

  20. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α.

    PubMed

    Kim, Hyung Gyun; Han, Eun Hee; Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo; Jeong, Hye Gwang

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. PMID:26296470

  1. Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress.

    PubMed

    Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P; Dobaria, Jentilal R

    2016-01-01

    Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity. PMID:27446163

  2. Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress

    PubMed Central

    Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P.; Dobaria, Jentilal R.

    2016-01-01

    Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity. PMID:27446163

  3. Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress.

    PubMed

    Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P; Dobaria, Jentilal R

    2016-01-01

    Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Yip1A, a novel host factor for the activation of the IRE1 pathway of the unfolded protein response during Brucella infection.

    PubMed

    Taguchi, Yuki; Imaoka, Koichi; Kataoka, Michiyo; Uda, Akihiko; Nakatsu, Daiki; Horii-Okazaki, Sakuya; Kunishige, Rina; Kano, Fumi; Murata, Masayuki

    2015-03-01

    Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments.  On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation. PMID:25742138

  15. Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic beta-cells.

    PubMed

    Veluthakal, Rajakrishnan; Madathilparambil, Suresh Vasu; McDonald, Phillip; Olson, Lawrence Karl; Kowluru, Anjaneyulu

    2009-01-01

    Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion (GSIS). However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic beta-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-induced, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation (i.e. GTP-bound form) and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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 rec