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Sample records for camp-regulated guanine nucleotide

  1. Signal transduction by guanine nucleotide binding proteins.

    Science.gov (United States)

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  2. Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes

    OpenAIRE

    Randazzo, Paul A.; Jian, Xiaoying; Chen, Pei-Wen; Zhai, Peng; Soubias, Olivier; Northup, John K.

    2014-01-01

    The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs), 1 15 Arf GEFs, 2 81 Rho GEFs, 3 8 Ras GEFs, 4 and others for other families of GTPases, 5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically. ...

  3. Analysis of Guanine Nucleotide Binding and Exchange Kinetics of the Escherichia coli GTPase Era

    OpenAIRE

    Sullivan, S M; Mishra, R.; Neubig, R. R.; Maddock, J. R.

    2000-01-01

    Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (N-methyl-3′-O-anthraniloyl) guanine nucleotide analogs. The equilibrium binding constants (KD) fo...

  4. Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

    OpenAIRE

    Hofer, F.; Fields, S; Schneider, C; Martin, G S

    1994-01-01

    The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The...

  5. Spatiotemporal regulation of Rap guanine nucleotide exchange factors

    NARCIS (Netherlands)

    Consonni, S.V.

    2014-01-01

    Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) orchestrate the activity of small G-proteins. In response to extracellular stimuli, GEFs and GAPs activate signaling cascades regulated by G-proteins by controlling their regulation in time and in space. Generally, GEFs

  6. Scambio, a novel guanine nucleotide exchange factor for Rho

    OpenAIRE

    Groffen John; Senadheera Dinithi; Haataja Leena; Hemmeryckx Bianca; Curtis Christina; Heisterkamp Nora

    2004-01-01

    Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is lo...

  7. Spatiotemporal regulation of Rap guanine nucleotide exchange factors

    OpenAIRE

    Consonni, S.V.

    2014-01-01

    Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) orchestrate the activity of small G-proteins. In response to extracellular stimuli, GEFs and GAPs activate signaling cascades regulated by G-proteins by controlling their regulation in time and in space. Generally, GEFs function as activators of G-proteins by promoting their GTP-bound state while GAPs serve as inhibitors by increasing the rate of GTP hydrolysis. Our understanding of the mechanisms of regulation o...

  8. Scambio, a novel guanine nucleotide exchange factor for Rho

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    Groffen John

    2004-04-01

    Full Text Available Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

  9. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

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    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  10. Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides.

    OpenAIRE

    Cai, Y. C.; Bullard, J. M.; Thompson, N L; Spremulli, L L

    2000-01-01

    Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to mea...

  11. Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

    OpenAIRE

    Hattori, S; Clanton, D J; Satoh, T.; Nakamura, S.; Kaziro, Y; Kawakita, M; Shih, T Y

    1987-01-01

    The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

  12. Kinetic Analysis of the Guanine Nucleotide Exchange Activity of TRAPP, a Multimeric Ypt1p Exchange Factor

    OpenAIRE

    Chin, Harvey F.; Cai, Yiying; Menon, Shekar; Ferro-Novick, Susan; Reinisch, Karin M; De La Cruz, Enrique M.

    2009-01-01

    The TRAPP complexes, large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEF) that activate the Rab GTPase Ypt1p. Here we measured the rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (GDP and GTP/GMPPNP) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic basis by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotid...

  13. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    International Nuclear Information System (INIS)

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125I-[Tyr1]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg2+. When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125I-[Tyr1]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

  14. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  15. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  16. Molecular Evolution of the Rab-Escort-Protein/Guanine-Nucleotide-Dissociation-Inhibitor Superfamily

    OpenAIRE

    Alory, Christelle; Balch, William E.

    2003-01-01

    Prenylation of Rab GTPases regulating vesicle traffic by Rab geranylgeranyltransferase (RabGGTase) requires a complex formed by the association of newly synthesized Rab proteins with Rab-escort-protein (REP), the choroideremia-gene-product that is mutated in disease, leading to loss of vision. After delivery to the membrane by the REP–Rab complex, subsequent recycling to the cytosol requires the REP-related guanine-nucleotide-dissociation-inhibitor (GDI). Although REP and GDI share common Rab...

  17. The emerging role of guanine nucleotide exchange factors in ALS and other neurodegenerative diseases

    OpenAIRE

    Droppelmann, Cristian A.; Campos-Melo, Danae; Volkening, Kathryn; Strong, Michael J.

    2014-01-01

    Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation, and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs), of which two classes: Dbl-related exchange factors and the more recently described dedicator of cytokinesis proteins family exchange factors. Increasingly, deregulation of normal GEF activity or function has been assoc...

  18. Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2

    OpenAIRE

    Arora, P. D.; Marignani, P A; McCulloch, C. A.

    2008-01-01

    Collagen phagocytosis is a crucial α2β1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5–20 min) but not Cdc42 or RhoA activity....

  19. Superoxide Inhibits Guanine Nucleotide Exchange Factor (GEF) Action on Ras, but not on Rho, through Desensitization of Ras to GEF

    OpenAIRE

    Wey, Michael; Phan, Vinh; Yepez, Gerardo; Heo, Jongyun

    2014-01-01

    Ras and Rho GTPases are molecular switches for various vital cellular signaling pathways. Overactivation of these GTPases often causes development of cancer. Guanine nucleotide exchange factors (GEFs) and oxidants function to upregulate these GTPases through facilitation of guanine nucleotide exchange (GNE) of these GTPases. However, the effect of oxidants on GEF functions, or vice versa, has not been known. We show that, via targeting Ras Cys51, an oxidant inhibits the catalytic action of Cd...

  20. Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras.

    OpenAIRE

    Park, W.; Mosteller, R D; Broek, D.

    1994-01-01

    Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF). Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid. We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae. Also, peptides corresponding to residues 1...

  1. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

    OpenAIRE

    Jones, S; Vignais, M L; Broach, J R

    1991-01-01

    The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also o...

  2. BIG2, A Guanine Nucleotide Exchange Factor for ADP-Ribosylation Factors: Its Localization to Recycling Endosomes and Implication in the Endosome IntegrityD⃞

    OpenAIRE

    Shin, Hye-Won; Morinaga, Naoko; NODA, Masatoshi; Nakayama, Kazuhisa

    2004-01-01

    Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is a...

  3. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    Energy Technology Data Exchange (ETDEWEB)

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. (Weizmann Institute, Rehovoth (Israel))

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  4. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    Science.gov (United States)

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  5. Hepatitis B virus X protein interacts with β5 subunit of heterotrimeric guanine nucleotide binding protein

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    Chen Wei

    2005-08-01

    Full Text Available Abstract Background To isolate cellular proteins interacting with hepatitis B virus X protein (HBX, from HepG2 cells infected with hepatitis B virus (HBV. Results HBV particles were produced in culture medium of HepG2 cells transfected with the mammalian expression vector containing the linear HBV genome, as assessed by commercially available ELISA assay. A cDNA library was made from these cells exposed to HBV. From yeast two hybrid screening with HBX as bait, human guanine nucleotide binding protein β subunit 5L (GNβ5 was isolated from the cDNA library constructed in this study as a new HBX-interacting protein. The HBX-GNβ5 interaction was further supported by mammalian two hybrid assay. Conclusion The use of a cDNA library constructed from HBV-transfected HepG2 cells has resulted in the isolation of new cellular proteins interacting with HBX.

  6. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    Science.gov (United States)

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  7. Molecular determinants of the interaction between coxsackievirus protein 3A and guanine nucleotide exchange factor GBF1.

    NARCIS (Netherlands)

    Wessels, E.; Duijsings, D.; Lanke, K.H.W.; Melchers, W.J.G.; Jackson, C.L.; Kuppeveld, F.J.M. van

    2007-01-01

    The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide

  8. Guanine nucleotide exchange factor H1 can be a new biomarker of melanoma

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    Shi, Jie; Guo, Bingyu; Zhang, Yu; Hui, Qiang; Chang, Peng; Tao, Kai

    2016-01-01

    Guanine nucleotide exchange factor H1 (GEF-H1), which couples microtubule dynamics to RhoA activation, is a microtubule-regulated exchange factor. Studies have shown that GEF-H1 can be involved in various cancer pathways; however, the clinical significance of GEF-H1 expression and functions in melanoma has not been established. In this study, we investigated the relationship between clinical outcomes and GEF-H1 functions in melanoma. A total of 60 cases of different grades of melanoma samples were used to detect the expression of GEF-H1. Results showed that both messenger RNA and protein levels of GEF-H1 were significantly higher in high-grade melanomas. Furthermore, patients with high GEF-H1 expression had a shorter overall survival (22 months) than patients with low level of GEF-H1 expression (33.38 months). We also found that GEF-H1 can promote the proliferation and metastasis of melanoma cells. In summary, these results suggested that GEF-H1 may be a valuable biomarker for assessing the degree and prognosis of melanoma following surgery.

  9. The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

    Science.gov (United States)

    Li, Hai-Sheng; Shome, Kuntala; Rojas, Raúl; Rizzo, Mark A; Vasudevan, Chandrasekaran; Fluharty, Eric; Santy, Lorraine C; Casanova, James E; Romero, Guillermo

    2003-01-01

    Background Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor. PMID:12969509

  10. Arf6 guanine-nucleotide exchange factor cytohesin-2 regulates myelination in nerves.

    Science.gov (United States)

    Torii, Tomohiro; Ohno, Nobuhiko; Miyamoto, Yuki; Kawahara, Kazuko; Saitoh, Yurika; Nakamura, Kazuaki; Takashima, Shou; Sakagami, Hiroyuki; Tanoue, Akito; Yamauchi, Junji

    2015-05-01

    In postnatal development of the peripheral nervous system (PNS), Schwann cells differentiate to insulate neuronal axons with myelin sheaths, increasing the nerve conduction velocity. To produce the mature myelin sheath with its multiple layers, Schwann cells undergo dynamic morphological changes. While extracellular molecules such as growth factors and cell adhesion ligands are known to regulate the myelination process, the intracellular molecular mechanism underlying myelination remains unclear. In this study, we have produced Schwann cell-specific conditional knockout mice for cytohesin-2, a guanine-nucleotide exchange factor (GEF) specifically activating Arf6. Arf6, a member of the Ras-like protein family, participates in various cellular functions including cell morphological changes. Cytohesin-2 knockout mice exhibit decreased Arf6 activity and reduced myelin thickness in the sciatic nerves, with decreased expression levels of myelin protein zero (MPZ), the major myelin marker protein. These results are consistent with those of experiments in which Schwann cell-neuronal cultures were treated with pan-cytohesin inhibitor SecinH3. On the other hand, the numbers of Ki67-positive cells in knockout mice and controls are comparable, indicating that cytohesin-2 does not have a positive effect on cell numbers. Thus, signaling through cytohesin-2 is required for myelination by Schwann cells, and cytohesin-2 is added to the list of molecules known to underlie PNS myelination. PMID:25824033

  11. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

    Science.gov (United States)

    Marei, Hadir; Carpy, Alejandro; Macek, Boris; Malliri, Angeliki

    2016-08-01

    The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects. PMID:27152953

  12. The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin.

    Directory of Open Access Journals (Sweden)

    K Farid Ahmad

    Full Text Available Intersectin-1L is a member of the Dbl homology (DH domain guanine nucleotide exchange factors (GEF which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.

  13. The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

    Directory of Open Access Journals (Sweden)

    Fluharty Eric

    2003-09-01

    Full Text Available Abstract Background Phospholipase D (PLD is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.

  14. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  15. Lpg0393 of Legionella pneumophila Is a Guanine-Nucleotide Exchange Factor for Rab5, Rab21 and Rab22

    Science.gov (United States)

    Sohn, Young-Sik; Shin, Ho-Chul; Park, Wei Sun; Ge, Jianning; Kim, Chan-Hee; Lee, Bok Luel; Do Heo, Won; Jung, Jae U.; Rigden, Daniel John; Oh, Byung-Ha

    2015-01-01

    Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella. PMID:25821953

  16. The Guanine Nucleotide Exchange Factor SWAP-70 Modulates the Migration and Invasiveness of Human Malignant Glioma Cells12

    OpenAIRE

    SEOL, HO JUN; Smith, Christian A.; Salhia, Bodour; Rutka, James T.

    2009-01-01

    The malignant glioma is the most common primary human brain tumor. Its tendency to invade away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Accordingly, the molecular pathogenesis of glioma invasion is currently under investigation. Previously, we examined a gene expression array database comparing human gliomas to nonneoplastic controls and identified several Rac guanine nucleotide exchange factors with differential expression. Here, we...

  17. Host-deficiency in Vav 2/3 guanine nucleotide exchange factors impairs tumor growth, survival, and angiogenesis in vivo

    OpenAIRE

    Brantley-Sieders, Dana M.; Zhuang, Guanglei; Vaught, David; Freeman, Tanner; Hwang, Yoonha; Hicks, Donna; Chen, Jin

    2009-01-01

    Vav guanine nucleotide exchange factors (GEFs) modulate changes in cytoskeletal organization through activation of Rho, Rac, and Cdc42 small GTPases. While Vav1 expression is restricted to the immune system, Vav2 and 3 are expressed in several tissues, including highly vascularized organs. Here, we provide the first evidence that Vav2 and Vav3 function within the tumor microenvironment to promote tumor growth, survival, and neovascularization. Host Vav2/3-deficiency reduced microvascular dens...

  18. RINL, Guanine Nucleotide Exchange Factor Rab5-Subfamily, Is Involved in the EphA8-Degradation Pathway with Odin

    OpenAIRE

    Hiroaki Kajiho; Shinichi Fukushima; Kenji Kontani; Toshiaki Katada

    2012-01-01

    The Rab family of small guanosine triphosphatases (GTPases) plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs). Ras and Rab interactor (or Ras interaction/interference)-like (RINL), which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that R...

  19. Expression of the wild-type p53 antioncogene induces guanine nucleotide-dependent stem cell division kinetics.

    OpenAIRE

    Sherley, J L; Stadler, P B; Johnson, D. R.

    1995-01-01

    The predominant type of cell division in adult mammals is renewal growth. Renewing stem cells in somatic tissues undergo continuous asymmetric divisions. One new daughter cell retains the division potential of the original stem cell, while the other differentiates into a functional constituent of the tissue. Disruptions of this process lead to the development of human cancers. We show that through a guanine nucleotide-dependent mechanism, the p53 antioncogene can induce exponentially dividing...

  20. Sestrins Function as Guanine Nucleotide Dissociation Inhibitors for Rag GTPases to Control mTORC1 Signaling

    OpenAIRE

    Peng, Min; Yin, Na; Li, Ming O.

    2014-01-01

    Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. Here we show that Sestrins bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino acid-induced...

  1. Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells

    OpenAIRE

    Sakabe Isamu; Asai Azusa; Iijima Junko; Maruyama Mitsuo

    2012-01-01

    Abstract Background We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cells after immunization with T-cell-dependent antigen. Zizimin2 was revealed to be a new family member of Dock (dedicator of cytokinesis), Dock11, which is the guanine nucleotide exchange factor for Cdc42, a low-molecular-weight GTPase. However, the molecular function of Zizimin2 in acquired immunity has not been elucidated. Results In this stud...

  2. Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Gromadski, Kirill B; Schümmer, Tobias; Strømgaard, Anne;

    2007-01-01

    of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows...... no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. Udgivelsesdato: 2007-Dec-7...

  3. Multisite phosphorylation of the guanine nucleotide exchange factor Cdc24 during yeast cell polarization.

    Directory of Open Access Journals (Sweden)

    Stephanie C Wai

    Full Text Available BACKGROUND: Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process. METHODOLOGY/PRINCIPAL FINDINGS: Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology. CONCLUSIONS/SIGNIFICANCE: We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.

  4. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Carla J Eaton

    Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

  5. Catching Functional Modes and Structural Communication in Dbl Family Rho Guanine Nucleotide Exchange Factors.

    Science.gov (United States)

    Raimondi, Francesco; Felline, Angelo; Fanelli, Francesca

    2015-09-28

    Computational approaches such as Principal Component Analysis (PCA) and Elastic Network Model-Normal Mode Analysis (ENM-NMA) are proving to be of great value in investigating relevant biological problems linked to slow motions with no demand in computer power. In this study, these approaches have been coupled to the graph theory-based Protein Structure Network (PSN) analysis to dissect functional dynamics and structural communication in the Dbl family of Rho Guanine Nucleotide Exchange Factors (RhoGEFs). They are multidomain proteins whose common structural feature is a DH-PH tandem domain deputed to the GEF activity that makes them play a central role in cell and cancer biology. While their common GEF action is accomplished by the DH domain, their regulatory mechanisms are highly variegate and depend on the PH and the additional domains as well as on interacting proteins. Major evolutionary-driven deformations as inferred from PCA concern the α6 helix of DH that dictates the orientation of the PH domain. Such deformations seem to depend on the mechanisms adopted by the GEF to prevent Rho binding, i.e. functional specialization linked to autoinhibition. In line with PCA, ENM-NMA indicates α6 and the linked PH domain as the portions of the tandem domain holding almost the totality of intrinsic and functional dynamics, with the α6/β1 junction acting as a hinge point for the collective motions of PH. In contrast, the DH domain holds a static scaffolding and hub behavior, with structural communication playing a central role in the regulatory actions by other domains/proteins. Possible allosteric communication pathways involving essentially DH were indeed found in those RhoGEFs acting as effectors of small or heterotrimeric RasGTPases. The employed methodology is suitable for deciphering structure/dynamics relationships in large sets of homologous or analogous proteins.

  6. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    OpenAIRE

    Price, S R; Nightingale, M.; Tsai, S C; Williamson, K. C.; Adamik, R; H. C. Chen; Moss, J; M. Vaughan

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on th...

  7. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    International Nuclear Information System (INIS)

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs

  8. Guanine-Centric Self-Assembly of Nucleotides in Water: An Important Consideration in Prebiotic Chemistry

    OpenAIRE

    Cassidy, Lauren M.; Burcar, Bradley T.; Stevens, Wyatt; Moriarty, Elizabeth M.; McGown, Linda B.

    2014-01-01

    Investigations of plausible prebiotic chemistry on early Earth must consider not only chemical reactions to form more complex products such as proto-biopolymers but also reversible, molecular self-assembly that would influence the availability, organization, and sequestration of reactant molecules. The self-assembly of guanosine compounds into higher-order structures and lyotropic liquid crystalline “gel” phases through formation of hydrogen-bonded guanine tetrads (G-tetrads) is one such cons...

  9. Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein.

    OpenAIRE

    Mukherjee, A; Dai, K; Lutkenhaus, J

    1993-01-01

    FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleoti...

  10. The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

    Directory of Open Access Journals (Sweden)

    Sarah V Consonni

    Full Text Available PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

  11. Influence of morphine on levels of type Ⅱ inhibitory guanine nucleotide binding protein in primary hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Qinghua Wu; Qiang Fu; Xinhua Wang; Jianhua Zhao; Liwei Liu; Shirong Tang

    2008-01-01

    BACKGROUND: The pharmacological action of opioid drugs is related to signal transduction of inhibitory guanine nucleotide binding protein.OBJECTIVE: To quantitatively and qualitatively analyze the influence of morphine on levels of type Ⅱ inhibitory guanine nucleotide binding protein (Gi2 protein) in primary cultured hippocampal neurons at different time points.DESIGN, TIME AND SETTING: A randomized controlled study, which was performed at the Department of Neurobiology, Changzheng Hospital, Second Military Medical University of Chinese PLA between September 2002 and March 2004.MATERIALS: Cerebral hippocampal neurons were obtained from newborn SD rats at 1-2 days of age. Biotin-antibody Ⅱ-avidin fluorescein isothiocyanate (Avidin-FITC) was purchased from Sigma Company (USA) and the Gi2 protein polyclonal antibody from Santa Cruz Biochemistry Company (USA).METHODS: Seven days after culture, mature hippocampal neurons were randomly divided into six groups: 4-, 8-, 16-, 24-, and 48-hour morphine groups, and a blank control group. Neurons in the morphine groups Received morphine (10μmol/L), which could cause alterations of G-protein mRNA and cAMP expression in the prefrontal cortex. Neurons in the blank control group were given the same volume of saline.MAIN OUTCOME MEASURES: Gi2 protein levels were detected by an immunofluorescence technique, and were analyzed by the image analytic system with the use of green fluorescence intensity.RESULTS: Gi2 protein levels in hippocampal neurons gradually decreased in the 4-, 8-, 16-, 24-, and 48-hour morphine groups. In particular, Gi2 protein levels in the 16-, 24-, and 48-hour morphine groups were significantly lower than that in the blank control group (P<0.05-0.01).CONCLUSION: Morphine may decrease Gi2 protein level in primary hippocampal neurons, and the decreasing trend is positively related to morphine-induced time.

  12. Guanine Nucleotide-Binding Proteins of the G(12) Family Shape Immune Functions by Controlling CD4(+) T Cell Adhesiveness and Motility

    NARCIS (Netherlands)

    S. Herroeder; P. Reichardt; A. Sassmann; B. Zimmermann; D. Jaeneke; J. Hoeckner; M.W. Hollmann; K.D. Fischer; S. Vogt; R. Grosse; N. Hogg; M. Gunzer; S. Offermanns; N. Wettschureck

    2009-01-01

    Integrin-mediated adhesion plays a central role in T cell trafficking and activation. Genetic inactivation of the guanine nucleotide-binding (G) protein alpha-subunits G alpha(12) and G alpha(13) resulted in an increased activity of integrin leukocyte-function-antigen-1 in murine CD4(+) T cells. The

  13. The Roles of Guanine Nucleotide Binding Proteins in Health and Disease

    Directory of Open Access Journals (Sweden)

    A.O. Ibegbu

    2011-02-01

    Full Text Available G-proteins are important mediators of cellular and tissue functions and are characterised by a recognition site for Guanine Triphosphate (GTP, Guanine Diphosphate (GDP and possess intrinsic GTPase activity. They play important roles in signal transduction responsible for cytoskeletal remodelling, cellular differentiation and vesicular transport. They are made up of three types namely, the small G-proteins, the sensors and the heterotrimeric G-proteins. The G-protein heterotrimers consist of G-alpha (G", G-beta (G$ and G-gamma (G( subunits. Each heterotrimeric G-protein have different subunits and the combination of these subunits define the specific role of each G -protein. The activation of G" subunits regulates the activity of effector enzymes and ion channels while G$( subunits function in the regulation of mitogen-activated protein kinase (MAP-kinase pathway. The G-protein-mediated signal transduction is important in the regulation of a cells morphological and physiological response to external stimuli. MAPKs are involved in the phosphorylation of transcription factors that stimulate gene transcription. G"s stimulates adenylate cyclase, thereby increasing cyclic adenosine monophosphate (cAMP leading to the phosphorylation and subsequent activation of Ca2+ channels. G proteins are involved in disease pathology through several mechanisms which interfere with the G protein activity. Other disease pathologies associated with abnormal mutations in G proteins can interfere with signal transduction pathways which may involve signal transmission that is either excessive, by augmentation o f G protein function, or insufficient, via inactivation of G proteins.

  14. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

    Directory of Open Access Journals (Sweden)

    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  15. Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

    Science.gov (United States)

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2014-01-01

    In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

  16. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.

    Science.gov (United States)

    Zhou, Wu; Li, Xiaobo; Premont, Richard T

    2016-05-15

    The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease. PMID:27182061

  17. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Directory of Open Access Journals (Sweden)

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  18. A molecular dynamics study of Hras-GTP and GDP complexes: The properties of water molecules around guanine nucleotide

    Science.gov (United States)

    Miyakawa, T.; Morikawa, R.; Takasu, M.; Sugimori, K.; Kawaguchi, K.; Saito, H.; Nagao, H.

    2013-02-01

    We study the structures of Hras-GTP and Hras-GDP complexes in water in order to investigate the mechanism of hydrolysis of GTP in the Hras-GTP complex. Understanding of the mechanism of hydrolysis of GTP in the Hras-GTP complex plays a key role in overcoming the human cancer. We performed molecular dynamics (MD) simulations of Hras-GTP complex and Hras-GDP complex in water using AMBER03 parameters and our calculated parameters around Mg2+. Using the trajectories of the MD simulations, we calculated the radial distribution functions of water molecules around the phosphorus atoms in guanine nucleotide in each complex. We also calculated the radius of the first hydration sphere, the averaged number of water molecules in the first hydration sphere, and the distribution of duration time of water molecules in the first hydration sphere. We also calculated the distribution of water molecules with respect to the angle around the PG in GTP and PB in GDP. It is suggested that the hydrolysis is triggered by water molecules attacking γ-phosphate from the direction rotated 35° to the O1B from the axis defined by PG and O3B.

  19. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    Science.gov (United States)

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

    2015-12-01

    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

  20. The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

    Science.gov (United States)

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-06-27

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. PMID:24821726

  1. The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane Recycling in Osteoclasts*

    Science.gov (United States)

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-01-01

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. PMID:24821726

  2. Total synthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin).

    OpenAIRE

    Sakmar, T P; Khorana, H G

    1988-01-01

    To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the alpha-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthet...

  3. Activation of Rac1 by the Guanine Nucleotide Exchange Factor Dck1 Is Required for Invasive Filamentous Growth in the Pathogen Candida albicans

    OpenAIRE

    Hope, Hannah; Bogliolo, Stéphanie; Arkowitz, Robert A; Bassilana, Martine

    2008-01-01

    Rho G proteins and their regulators are critical for cytoskeleton organization and cell morphology in all eukaryotes. In the opportunistic pathogen Candida albicans, the Rho G proteins Cdc42 and Rac1 are required for the switch from budding to filamentous growth in response to different stimuli. We show that Dck1, a protein with homology to the Ced-5, Dock180, myoblast city family of guanine nucleotide exchange factors, is necessary for filamentous growth in solid media, similar to Rac1. Our ...

  4. Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

    Science.gov (United States)

    Mahajan, Divyanshu; Boh, Boon Kim; Zhou, Yan; Chen, Li; Cornvik, Tobias Carl; Hong, Wanjin; Lu, Lei

    2013-11-01

    Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

  5. The t-complex-encoded guanine nucleotide exchange factor Fgd2 reveals that two opposing signaling pathways promote transmission ratio distortion in the mouse

    OpenAIRE

    Bauer, Hermann; Véron, Nathalie; Willert, Jürgen; Herrmann, Bernhard G.

    2007-01-01

    Transmission ratio distortion (TRD), the preferential inheritance of the t haplotype from t/+ males, is caused by the cooperative effect of four t-complex distorters (Tcd1–4) and the single t-complex responder (Tcr) on sperm motility. Here we show that Fgd2, encoding a Rho guanine nucleotide exchange factor, maps to the Tcd2 region. The t allele of Fgd2 is overexpressed in testis compared with wild type. A loss-of-function allele of Fgd2 generated by gene targeting reduces the transmission ra...

  6. Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A

    OpenAIRE

    Cans, Christophe; Passer, Brent J.; Shalak, Vyacheslav; Nancy-Portebois, Vanessa; Crible, Virginie; Amzallag, Nathalie; Allanic, David; Tufino, Rowena; Argentini, Manuela; Moras, Dino; Fiucci, Giusy; Goud, Bruno; Mirande, Marc; Amson, Robert; Telerman, Adam

    2003-01-01

    Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bβ, as TCTP-interacting partners. In vitro and i...

  7. Unmasking of magnesium-dependent high-affinity binding sites for [dAla2, dLeu5]enkephalin after pretreatment of brain membranes with guanine nucleotides.

    OpenAIRE

    Chang, K.J.; Blanchard, S G; Cuatrecasas, P

    1983-01-01

    The regulation of mu- and delta-opiate receptors by guanine nucleotides and cations was studied by examining the binding of [3H][DAla2, DLeu5]enkephalin to rat brain membranes. The binding to mu-opiate receptors could be suppressed by 1 microM [DPro4]morphiceptin, a highly specific mu-agonist, thus permitting separate assessment of delta-opiate receptor binding. GTP, GDP, and the nonhydrolyzable analogs 5'-guanylyl imidodiphosphate (Gpp[NH]p) and guanosine 5'-O-(2-thiodiphosphate) (GDP-S) eff...

  8. eIF2B promotes eIF5 dissociation from eIF2•GDP to facilitate guanine nucleotide exchange for translation initiation

    OpenAIRE

    Jennings, Martin D.; Zhou, Yu; Mohammad-Qureshi, Sarah S.; Bennett, David; Pavitt, Graham D.

    2013-01-01

    Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-activating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). Jennings et al. now establish a second activity for eIF2B (as a GDI displacement factor [GDF]) and demonstrate that this function is independent of its GEF activity. This study suggests that eIF2B is a bifunctional protein and def...

  9. Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

    OpenAIRE

    Divyanshu Mahajan; Boon Kim Boh; Yan Zhou; Li Chen; Tobias Carl Cornvik; Wanjin Hong; Lei Lu

    2013-01-01

    Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or ce...

  10. GBF1, a guanine nucleotide exchange factor for Arf, is crucial for coxsackievirus B3 RNA replication.

    NARCIS (Netherlands)

    Lanke, K.H.W.; Schaar, H.M. van der; Belov, G.A.; Feng, Q.; Duijsings, D.; Jackson, C.L.; Ehrenfeld, E.; Kuppeveld, F.J.M. van

    2009-01-01

    The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show

  11. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    Energy Technology Data Exchange (ETDEWEB)

    Carlow, D.A.; Teh, H.S.; Marth, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  12. Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

    DEFF Research Database (Denmark)

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea;

    2015-01-01

    . More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation...... of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to...... activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase...

  13. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. (Fitzsimons Army Medical Center, Aurora, CO (USA))

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  14. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    International Nuclear Information System (INIS)

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G-3H] hypoxanthine (Hy) into [3H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [3H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation

  15. The Bipartite Rac1 Guanine Nucleotide Exchange Factor Engulfment and Cell Motility 1/Dedicator of Cytokinesis 180 (Elmo1/Dock180) Protects Endothelial Cells from Apoptosis in Blood Vessel Development

    NARCIS (Netherlands)

    Schaeker, Kathrin; Bartsch, Susanne; Patry, Christian; Stoll, Sandra J.; Hillebrands, Jan-Luuk; Wieland, Thomas; Kroll, Jens

    2015-01-01

    Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmol/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Racl). Elmol/Dock180 regulates Racl activity in a specific spatiotemporal manner in endothelial cells (EC

  16. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration–proliferation dichotomy

    Science.gov (United States)

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L.; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

    2015-01-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously—a phenomenon called “migration–proliferation dichotomy.” We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration–proliferation dichotomy during cancer invasion, wound healing, and development. PMID:26286990

  17. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration-proliferation dichotomy.

    Science.gov (United States)

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L; Garcia-Marcos, Mikel; Farquhar, Marilyn G; Ghosh, Pradipta

    2015-09-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development. PMID:26286990

  18. Distinct subcellular localization of alternative splicing variants of EFA6D, a guanine nucleotide exchange factor for Arf6, in the mouse brain.

    Science.gov (United States)

    Fukaya, Masahiro; Ohta, Shingo; Hara, Yoshinobu; Tamaki, Hideaki; Sakagami, Hiroyuki

    2016-09-01

    EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc. PMID:27241101

  19. Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge.

    Science.gov (United States)

    Biersmith, Bridget; Wang, Zong-Heng; Geisbrecht, Erika R

    2015-06-01

    The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to engulfment and cell motility (ELMO) proteins, Dock-ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes, including cell motility, apoptotic cell clearance, and axon guidance. Overlapping expression patterns and functional redundancy among the 11 vertebrate Dock family members, which are subdivided into four families (Dock A, B, C, and D), complicate genetic analysis. In both vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism for understanding Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock A) and Sponge (Spg; Dock B). Here we show that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel. Moreover, we confirm the in vivo role of Mbc upstream of Rac and provide evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. Our findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis.

  20. Distinct Functions for Arf Guanine Nucleotide Exchange Factors at the Golgi Complex: GBF1 and BIGs Are Required for Assembly and Maintenance of the Golgi Stack and trans-Golgi Network, Respectively

    OpenAIRE

    Manolea, Florin; Claude, Alejandro; Chun, Justin; Rosas, Javier; Melançon, Paul

    2008-01-01

    We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, whereas BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the endoplasmic reti...

  1. Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

    Energy Technology Data Exchange (ETDEWEB)

    Bielnicki, Jakub A.; Shkumatov, Alexander V.; Derewenda, Urszula; Somlyo, Avril V.; Svergun, Dmitri I.; Derewenda, Zygmunt S. (EMBL); (UV)

    2012-10-09

    PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via G{alpha}{sub 12/13} and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory 'activation box' and the 'GEF switch,' which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

  2. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  3. The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

    Directory of Open Access Journals (Sweden)

    Melissa C Srougi

    Full Text Available Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs, Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

  4. The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells

    Institute of Scientific and Technical Information of China (English)

    Jie YIN; Ya-juan WAN; Shi-yang LI; Ming-juan DU; Cui-zhu ZHANG; Xing-long ZHOU; You-jia CAO

    2011-01-01

    Aim: To investigate a novel function of proto-oncogene Vavl in the apoptosis of human leukemia Jurkat cells.Methods: Jurkat cells,Jurkat-derived vavl-null cells(J.Vavl)and Vavl-reconstituted J.WT cells were treated with a Fas agonist antibody,IgM clone CH11.Apoptosis was determined using propidium iodide(PI)staining,Annexin-V staining,DNA fragmentation,cleavage of caspase 3/caspase 8,and poly(ADP-ribose)polymerase(PARP).Mitochondria transmembrane potential(Δψm)was measured using DiOC6(3)staining.Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot,respectively.Bcl-2 promoter activity was analyzed using luciferase reporter assays.Results: Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells.J.Vav1 cells lost mitochondria transmembrane potential(Δψm)more rapidly upon Fas induction.These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells.The expression of Vav1 increased the transcription of pro-survival Bcl-2.The guanine nucleotide exchange activity of Vav1was required for enhancing Bcl-2 promoter activity,and the Vav1 downstream substrate,small GTPase Rac2,was likely involved in the control of Bcl-2 expression.Conclusion: Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.

  5. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    Science.gov (United States)

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M

    2016-05-20

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  6. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  7. Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers.

    Science.gov (United States)

    Holland, Joseph G; Malin, Jessica N; Jordan, David S; Morales, Esmeralda; Geiger, Franz M

    2011-03-01

    This article reports nonlinear optical measurements that quantify, for the first time directly and without labels, how many Mg(2+) cations are bound to DNA 21-mers covalently linked to fused silica/water interfaces maintained at pH 7 and 10 mM NaCl, and what the thermodynamics are of these interactions. The overall interaction of Mg(2+) with adenine, thymine, guanine, and cytosine is found to involve -10.0 ± 0.3, -11.2 ± 0.3, -14.0 ± 0.4, and -14.9 ± 0.4 kJ/mol, and nonspecific interactions with the phosphate and sugar backbone are found to contribute -21.0 ± 0.6 kJ/mol for each Mg(2+) ion bound. The specific and nonspecific contributions to the interaction energy of Mg(2+) with oligonucleotide single strands is found to be additive, which suggests that within the uncertainty of these surface-specific experiments, the Mg(2+) ions are evenly distributed over the oligomers and not isolated to the most strongly binding nucleobase. The nucleobases adenine and thymine are found to bind only three Mg(2+) ions per 21-mer oligonucleotide, while the bases cytosine and guanine are found to bind eleven Mg(2+) ions per 21-mer oligonucleotide.

  8. GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Dong-Jie Jia; Xi Cao; Wei Wang; Xiao-Yun Tan; Xue-Qin Zhang; Li-Qun Chen; De Ye

    2009-01-01

    In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2.1 (gnl2-1) and gn12-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking, it was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.

  9. Biological Function of the Guanine Nucleotide Exchange Factor C3G%鸟嘌呤核苷酸交换因子C3G生物学功能研究进展

    Institute of Scientific and Technical Information of China (English)

    吴国金; 安输; 杨洋; 刘莹; 徐天瑞; 郭晓汐

    2016-01-01

    Crk SH3域结合鸟嘌呤核苷酸交换因子[v-Crk sarcoma virus CT10 oncogene homolog (avian) SH3-domain-binding guanine nucleotide exchange factor,C3G]属于鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factors,GEFs),是一种在细胞内普遍表达的蛋白质分子.C3G激活小GTP酶并参与各种信号所触发的通路,对哺乳动物胚胎发育和许多组织的细胞功能来说,都是必不可少的.C3G参与细胞的黏附和迁移,细胞连接的维持,突起的生长等需要细胞骨架重构重要生物功能的调控.在细胞信号转导通路中,C3G的功能不仅依赖其催化活性,更重要的是,C3G在不同细胞类型中与不同的蛋白质相互作用,从而激活相应的信号转导通路.该文总结了目前C3G研究的最新进展,并概述了C3G的不同表达亚型对癌症、心脏病的影响及其生物功能背后的分子作用机制.对C3G及其相互作用蛋白质的研究将为卵巢癌、心脏病等疾病的诊断和治疗提供重要的科学依据.

  10. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

    Directory of Open Access Journals (Sweden)

    Zhi Liu

    Full Text Available The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue-DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of

  11. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte;

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransfe......Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we.......5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase....

  12. An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

    International Nuclear Information System (INIS)

    The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs

  13. Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif.

    Science.gov (United States)

    Kimple, Randall J; Willard, Francis S; Hains, Melinda D; Jones, Miller B; Nweke, Gift K; Siderovski, David P

    2004-03-15

    GoLoco ('Galpha(i/o)-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Galpha subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Galpha(i1) subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Galpha(i1). Only the first and third GoLoco motifs within G18 are capable of interacting with Galpha subunits, and these bind with low micromolar affinity only to Galpha(i1) in the GDP-bound form, and not to Galpha(o), Galpha(q), Galpha(s) or Galpha12. Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Galpha and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Galpha binding and GDI activity. PMID:14656218

  14. Leukemia-associated Rho guanine-nucleotide exchange factor is not critical for RhoA regulation, yet is important for platelet activation and thrombosis in mice

    OpenAIRE

    Christopher M Williams; Harper, Matthew T.; Goggs, Robert A N; Walsh, Tony G.; Offermanns, S.; Poole, Alastair W.

    2015-01-01

    Summary Background RhoA is an important regulator of platelet responses downstream of Gα13, yet we still know little about its regulation in platelets. Leukemia‐associated Rho guanine‐nucleotide exchange factor (GEF [LARG]), a RhoA GEF, is highly expressed in platelets and may constitute a major upstream activator of RhoA. To this end, it is important to determine the role of LARG in platelet function and thrombosis. Methods and results Using a platelet‐specific gene knockout, we show that th...

  15. Establishment of lung cancer cell strain for stable expression of RAS guanine nucleotide releasing factor 2%稳定表达RAS鸟苷酸释放因子2肺癌细胞株的建立

    Institute of Scientific and Technical Information of China (English)

    王勇; 陈虹; 卜友泉

    2012-01-01

    Objective To construct a eukaryotic expression vector for RAS guanine nucleotide releasing factor 2 (RASGR-F2), tagged with GFP, and establish a lung cell strain for stable expression of RASGRF2. Methods Plasmid RASGRF2-pCMV6-Myc-DDK was digested with Sgf I and Not I , and RASGRF2 gene fragment was recovered and subcloned into vector pCMV6-GFP. Lung cancer HI299 cells were transfected with the constructed recombinant plasmid RASGRF2-pCMV6-GFP and observed for location of RASGRF2 protein under invert fluorescent microscope. The cell strain for stable expression of RASGRF2 was established by G418 screening, and the expression of RASGRF2 was determined by RT-PCR and Western blot. Results Restriction analysis and sequencing proved that RASGRF2 gene was successfully cloned into eukaryotic expression vector pCMV6-GFP. Recombinant RASGRF2-GFP protein was mainly expressed in cytoplasm of HI299 cells. RT-PCR and Western blot showed that RASGRF2 was stably expressed in HI299 cells. Conclusion The lung cancer cell strain for stable expression of RASGRF2 gene was successfully established, which laid a foundation of further study on function of RASGRF2 gene.%目的 构建带有GFP标签的RAS鸟苷酸释放因子2(RAS guanine nucleotide releasing factor 2,RASGRF2)真核表达质粒,并建立稳定表达RASGRF2的肺癌细胞株.方法 用Sgf Ⅰ和Not Ⅰ双酶切RASGRF2-pCMV6-Myc-DDK质粒,回收RASGRF2基因片段,亚克隆入pCMV6-GFP载体中,构建重组表达质粒RASGRF2-pCMV6-GFP,转染肿瘤细胞H1299,倒置荧光显微镜下观察RASGRF2蛋白的定位.经G418筛选并建立稳定表达RASGRF2的细胞株,RT-PCR及Western blot法检测RASGRF2的表达.结果 经双酶切及测序证实RASGRF2基因成功克隆至真核表达载体pCMV6-GFP中;重组RASGRF2-GFP蛋白在H1299细胞中主要在胞质中表达;经RT-PCR及Western blot证实,RASGRF2在H1299细胞中稳定表达.结论 成功建立了稳定表达RASGRF2基因的肺癌细胞株,为进一步研究RASGRF2

  16. EPR spectroelectrochemical investigation of guanine radical formation and environment effects

    OpenAIRE

    C. Ribaut; Bordeau, G.; Perio, Pierre; Reybier, K; Sartor, V.; Reynes, O.; Fabre, P. L.; Chouini-Lalanne, N.

    2014-01-01

    Guanine radical detection was carried out by a new convenient and efficient method coupling electron paramagnetic resonance spectroscopy and indirect electro-oxidation of guanine in different biological environments, from the free nucleotide to several types of DNA substrates. Compared to the widely used photoirradiation method, this method appeared more selective in the choice of the electrochemical mediator. Carried out in presence of a ruthenium mediator and PBN as spin trap, this method r...

  17. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei

    OpenAIRE

    Schaffitzel, Christiane; Berger, Imre; Postberg, Jan; Hanes, Jozef; Lipps, Hans J; Plückthun, Andreas

    2001-01-01

    Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function...

  18. PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling.

    Science.gov (United States)

    Liu, Heng-Jia; Ooms, Lisa M; Srijakotre, Nuthasuda; Man, Joey; Vieusseux, Jessica; Waters, JoAnne E; Feng, Yue; Bailey, Charles G; Rasko, John E J; Price, John T; Mitchell, Christina A

    2016-08-12

    PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21(WAF1/CIP1) PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stable PREX1 knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression. PMID:27358402

  19. Chlamydomonas reinhardtii telomere repeats form unstable structures involving guanine-guanine base pairs.

    OpenAIRE

    Petracek, M E; Berman, J.

    1992-01-01

    Unusual DNA structures involving four guanines in a planar formation (guanine tetrads) are formed by guanine-rich (G-rich) telomere DNA and other G-rich sequences (reviewed in (1)) and may be important in the structure and function of telomeres. These structures result from intrastrand and/or interstrand Hoogsteen base pairs between the guanines. We used the telomeric repeat of Chlamydomonas reinhardtii, TTTTAGGG, which contains 3 guanines and has a long interguanine A + T tract, to determine...

  20. Endogenous melatonin and oxidatively damaged guanine in DNA

    Directory of Open Access Journals (Sweden)

    Poulsen Henrik E

    2009-10-01

    Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

  1. Small guanine nucleotide-binding protein Rho and myocardial function

    Institute of Scientific and Technical Information of China (English)

    Jun REN; Cindy X FANG

    2005-01-01

    RhoA and Rho-kinase (ROCK) participate in a wide variety of cell signal functions such as cell growth, smooth and cardiac muscle contraction, cytoskeleton rearrangement, cell migration and proliferation. In vascular smooth muscle cells,RhoA and ROCK play an important role in Ca2+ sensitization and regulate vascular smooth muscle tone. In the heart, RhoA and ROCK mediate hypertrophic response leading to cardiac hypertrophy. Recent cellular and molecular biology studies using ROCK inhibitors such as Y-27632 and fasudil have indicated a pivotal role of the RhoA-ROCK cascade in many aspects of cardiovascular function such as cardiac hypertrophy and ventricular remodeling following myocardial infarction. Inhibition of the RhoA-ROCK signaling pathway may be a suitable target for a number of cardiovascular diseases including hypertension, atherosclerosis, diabetes and hypertrophic heart failure. This review focuses on the current understanding of the RhoA-ROCK signal pathway in heart diseases and discusses the use of ROCK inhibitors as therapeutic agents for heart diseases ranging from hypertensive cardiomyopathy to heart failure.

  2. Nucleotide Capacitance Calculation for DNA Sequencing

    OpenAIRE

    Lu, Jun-Qiang; Zhang, X.-G.

    2008-01-01

    Using a first-principles linear response theory, the capacitance of the DNA nucleotides, adenine, cytosine, guanine, and thymine, are calculated. The difference in the capacitance between the nucleotides is studied with respect to conformational distortion. The result suggests that although an alternate current capacitance measurement of a single-stranded DNA chain threaded through a nanogap electrode may not be sufficient to be used as a standalone method for rapid DNA sequencing, the capaci...

  3. Preparation and bioevaluation of 99mTc-carbonyl complex of guanine

    International Nuclear Information System (INIS)

    The aim of this study is to prepare radiolabeled guanine with 99mTc(CO)3+ core. For this purpose, guanine has been radiolabeled with 99mTc(CO)3+ core. Quality control study of radiolabeled guanine molecule with 99mTc(CO)3+ core was performed by thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC). The results showed that the radiolabeling yield was quite high (94 ± 3%). Beside that 99mTc(CO)3-Gua complex has showed good in vitro stability during the 24 h period. Radiopharmaceutical potential of this complex was evaluated in Wistar Albino Rats. It was concluded that 99mTc(CO)3-Gua could be used as a nucleotide radiopharmaceutical for in vivo applications. (author)

  4. Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release

    DEFF Research Database (Denmark)

    Leonardsen, L; DeClue, J E; Lybaek, H;

    1996-01-01

    , the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity...

  5. Dictyostelium Ric8 is a nonreceptor guanine exchange factor for heterotrimeric G proteins and is important for development and chemotaxis

    NARCIS (Netherlands)

    Kataria, Rama; Xu, Xuehua; Fusetti, Fabrizia; Keizer-Gunnink, Ineke; Jin, Tian; van Haastert, Peter J M; Kortholt, Arjan

    2013-01-01

    Heterotrimeric G proteins couple external signals to the activation of intracellular signal transduction pathways. Agonist-stimulated guanine nucleotide exchange activity of G-protein-coupled receptors results in the exchange of G-protein-bound GDP to GTP and the dissociation and activation of the c

  6. The Emerging Role of Guanine Exchange Factors in ALS and other neurodegenerative diseases

    OpenAIRE

    Cristian eDroppelmann; Danae eCampos-Melo; Kathryn eVolkening; Michael Joseph Strong

    2014-01-01

    Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs), of which two classes: Dbl-related exchange factors and the more recently described Dock family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of di...

  7. Chemisorption of Guanine on Cu(110)

    Science.gov (United States)

    Matos, Jeronimo; Kara, Abdelkader

    2011-03-01

    We use density functional theory (PBE) to calculate the adsorption of a guanine molecule on Cu(110). At saturation coverage, guanine adsorbs tilted with the oxygen atom strongly bound to one of the surface atoms at a height of 2.12 Å above this surface atom with a binding energy of 430 meV/molecule. The substrate top layer atoms show a buckling of 0.22 Å , while the molecule experiences a twist from the flat configuration in the gas phase. The dz 2 state of the copper atom -that is bound to the oxygen atom- presents an enhancement in its density near the Fermi level. We calculated a drop in the work function of 0.34 eV upon adsorption of guanine on Cu(110). These effects classify this system as chemisorption.

  8. Nucleotide Metabolism

    DEFF Research Database (Denmark)

    Martinussen, Jan; Willemoës, M.; Kilstrup, Mogens

    2011-01-01

    Metabolic pathways are connected through their utilization of nucleotides as supplier of energy, allosteric effectors, and their role in activation of intermediates. Therefore, any attempt to exploit a given living organism in a biotechnological process will have an impact on nucleotide metabolism....... The aim of this article is to provide knowledge of nucleotide metabolism and its regulation to facilitate interpretation of data arising from genetics, proteomics, and transcriptomics in connection with biotechnological processes and beyond....

  9. Identification of small molecule compounds with higher binding affinity to guanine deaminase (cypin) than guanine

    OpenAIRE

    Fernández, José R.; Sweet, Eric S; Welsh, William J.; Firestein, Bonnie L

    2010-01-01

    Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transp...

  10. Formation of guanine ribonucleotidyl-(3'-5')-adenosine in a flavinogenic strain of Eremothecium ashbyii.

    Science.gov (United States)

    Mitsuda, H; Nishikawa, Y; Nakajima, K

    1976-01-01

    The addition of caffeine caused the accumulation of a new nucleotide compound simultaneously with the rigid inhibition of ribofalvin production in non-growing cells of Eremothecium ashbyii. In the present study we tried to identify the structure of the nucleotide compound using non-growing cells of the mold. 1) It became possible to obtain a large amount of mycelia by masscultivation in a reagent tank. 2) A new nucleotide compound, referred to as compound A in the paper, was extracted with perchloric acid solution and purified by the following subsequent procedures: 1) Dowex 1 x 2 (HCOO-) column, 2) charcoal treatment, 3) DEAE-Sephadex A25 (CI-) column, 4) Dowex 1 x 2 (C1-) column, and 5) DEAE-Sephadex A25 (HCO3-) column. 3) The structure of the new nucleotide compound was proved to be guanine ribonucleotidyl-(3'-5')-adenosine (GpA) from the results of the following analyses: 1) alkaline degradation, 2) UV-spectra, IR-spectra and NMR-spectra, and 3) enzymatic treatments with RNase T2 and phosphodiesterase. 4) The roles of caffeine and guanine ribonucleotidyl-(3'-5')-adenosine in connection with flavinogenesis of this mold were discussed. PMID:182940

  11. Regulation of Salmonella typhimurium pyr Gene Expression: Effect of Changing Both Purine and Pyrimidine Nucleotide Pools

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1989-01-01

    permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly...

  12. The CBS subdomain of inosine 5’-monophosphate dehydrogenase regulates purine nucleotide turnover†

    OpenAIRE

    Pimkin, Maxim; Markham, George D.

    2008-01-01

    Inosine 5’-monophosphate dehydrogenase (IMPDH) catalyzes the rate limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP...

  13. Fluorescence enhancement of DNA-silver nanoclusters from guanine proximity

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsin-chih [Los Alamos National Laboratory; Sharma, Jaswinder [Los Alamos National Laboratory; Yoo, Hyojong [Los Alamos National Laboratory; Martinez, Jennifer S [Los Alamos National Laboratory

    2010-01-01

    Oligonucleotide-templated, silver nanoclusters (DNA/Ag NCs) are a versatile set of fluorophores and have already been used for live cell imaging, detection of specific metal ions, and single-nucleotide variation identification. Compared to commonly used organic dyes, these fluorescent nanoclusters have much better photostability and are often a few times brighter. Owing to their small size, simple preparation, and biocompatibility (i.e. made of nontoxic metals), DNA/Ag NCs should find more applications in biological imaging and chemical detection in the years to come. While clearly promising as new fluorophores, DNA/Ag NCs possess a unique and poorly understood dynamic process not shared by organic dyes or photoluminescent nanocrystals - the conversion among different NC species due to silver oxidation/reduction or NC regrouping. While this environmental sensitivity can be viewed as a drawback, in the appropriate context, it can be used as a sensor or reporter. Often reversible, conversions among different NC species have been found to depend upon a number of factors, including time, temperature, oxygen and salt content. In this communication, we report significant fluorescence enhancement of DNA/Ag NCs via interactions with guanine-rich DNA sequences. Moreover, we demonstrated this property can be used for sensitive detection of specific target DNA from a human oncogene (i.e. Braf gene).

  14. IR spectra of guanine and hypoxanthine isolated molecules

    Science.gov (United States)

    Sheina, G. G.; Stepanian, S. G.; Radchenko, E. D.; Blagoi, Yu. P.

    1987-05-01

    High resolution spectra of guanine, hypoxanthine, isocytosine, 2-aminopyrimidine and their deutero- and methyl derivatives obtained in Ar matrices by the low temperature IR spectroscopy method are reported. Normal modes of enol tautomers of guanine, 9-CH 3-guanine, hypoxanthine and 2-aminopurine are calculated. Force fields are the same as for purine. Results calculated are used to interpret the experimental spectra. Keto—enol tautomerism is shown to exist in guanine and hypoxanthine, the proportions of enol tautomer being 50 and 5%, respectively. Possible biological applications of the results obtained are discussed.

  15. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Michael Fasullo

    2015-04-01

    Full Text Available Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases.

  16. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Hazelton, Keith Z. [Yeshiva Univ., New York, NY (United States); Ho, Meng-Chaio [Yeshiva Univ., New York, NY (United States); Cassera, Maria B. [Yeshiva Univ., New York, NY (United States); Clinch, Keith [Industrial Research Ltd., Lower Hutt (New Zealand); Crump, Douglas R. [Industrial Research Ltd., Lower Hutt (New Zealand); Rosario Jr., Irving [Yeshiva Univ., New York, NY (United States); Merino, Emilio F. [Yeshiva Univ., New York, NY (United States); Almo, Steve C. [Yeshiva Univ., New York, NY (United States); Tyler, Peter C. [Industrial Research Ltd., Lower Hutt (New Zealand); Schramm, Vern L. [Yeshiva Univ., New York, NY (United States)

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  17. The Emerging Role of Guanine Exchange Factors in ALS and other neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    Cristian eDroppelmann

    2014-09-01

    Full Text Available Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs, of which two classes: Dbl-related exchange factors and the more recently described Dock family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF in the pathogenesis of amyotrophic lateral sclerosis (ALS. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament (NEFL mRNA 3’UTR to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.

  18. Cyclic nucleotide regulation of cardiac sympatho-vagal responsiveness.

    Science.gov (United States)

    Li, Dan; Paterson, David J

    2016-07-15

    Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are now recognized as important intracellular signalling molecules that modulate cardiac sympatho-vagal balance in the progression of heart disease. Recent studies have identified that a significant component of autonomic dysfunction associated with several cardiovascular pathologies resides at the end organ, and is coupled to impairment of cyclic nucleotide targeted pathways linked to abnormal intracellular calcium handling and cardiac neurotransmission. Emerging evidence also suggests that cyclic nucleotide coupled phosphodiesterases (PDEs) play a key role limiting the hydrolysis of cAMP and cGMP in disease, and as a consequence this influences the action of the nucleotide on its downstream biological target. In this review, we illustrate the action of nitric oxide-CAPON signalling and brain natriuretic peptide on cGMP and cAMP regulation of cardiac sympatho-vagal transmission in hypertension and ischaemic heart disease. Moreover, we address how PDE2A is now emerging as a major target that affects the efficacy of soluble/particulate guanylate cyclase coupling to cGMP in cardiac dysautonomia. PMID:26915722

  19. Experimental observation of guanine tautomers with VUV photoionization

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jia; Kostko, Oleg; Nicolas, Christophe; Tang, Xiaonan; Belau, Leonid; de Vries, Mattanjah S.; Ahmed, Musahid

    2008-12-01

    Two methods of preparing guanine in the gas phase, thermal vaporization and laser desorption, have been investigated. The guanine generated by each method is entrained in a molecular beam, single photon ionized with tunable VUV synchrotron radiation, and analyzed using reflectron mass spectrometry. The recorded photoionization efficiency (PIE) curves show a dramatic difference for experiments performed via thermal vaporization compared to laser desorption. The calculated vertical and adiabatic ionization energies for the eight lowest lying tautomers of guanine suggest the experimental observations arise from different tautomers being populated in the two different experimental methods.

  20. Exploring the characterization tools of Guanine-Quadruplexes.

    Science.gov (United States)

    Kaushik, Mahima; Kaushik, Shikha; Kukreti, Shrikant

    2016-01-01

    Occurrence of guanine-rich sequences throughout the genome at specific locations like chromosomal ends (telomeres), promoters and Untranslated regions (UTR's) is very well documented. Quite recently, visualization of guanine-quadruplex in human and mammalian cells have also provided a very significant evidence for the in vivo existence of guanine-quadruplex, reconfirming their biological relevance in cellular processes like replication, transcription, recombination, etc. Guanine quadruplexes have enormous potential of exhibiting various topologies which differ, by number/ orientation of strands or loop orientations etc. Some relatively new polymorphic structures like 3+1 quadruplex, G-triplex, and Tri-G-quadruplex have also been proposed for the guanine-rich sequences. Various biochemical and biophysical techniques have been used to characterize these multistranded DNA structures. An extensive review of the mechanistic models of the already existing and newly emerging techniques is actually required, which may further facilitate our understanding about these structures. This review aims to summarize some of these techniques along with their requirements and limitations, which might further give some insights for the fine tuning of the solution and environmental conditions needed for facilitating guanine-quadruplex formation. PMID:26709787

  1. The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth

    OpenAIRE

    Leo Chang; Tabita Kreko-Pierce; Eaton, Benjamin A.

    2015-01-01

    ABSTRACT The generation of neuronal morphology requires transport vesicles originating from the Golgi apparatus (GA) to deliver specialized components to the axon and dendrites. Drosophila Arfaptin is a membrane-binding protein localized to the GA that is required for the growth of the presynaptic nerve terminal. Here we provide biochemical, cellular and genetic evidence that the small GTPase Arl1 and the guanine-nucleotide exchange factor (GEF) Gartenzwerg are required for Arfaptin function ...

  2. Characterization of oxidative guanine damage and repair in mammalian telomeres.

    Directory of Open Access Journals (Sweden)

    Zhilong Wang

    2010-05-01

    Full Text Available 8-oxo-7,8-dihydroguanine (8-oxoG and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1-initiated DNA base excision repair (BER. Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH, by chromosome orientation-FISH (CO-FISH, and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/- mouse tissues and primary embryonic fibroblasts (MEFs cultivated in hypoxia condition (3% oxygen, whereas telomere shortening was detected in Ogg1(-/- mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/- mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/- mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/- MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity

  3. Guanine holes are prominent targets for mutation in cancer and inherited disease.

    Directory of Open Access Journals (Sweden)

    Albino Bacolla

    Full Text Available Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials. Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

  4. Reactivity of chitosan derivatives and their interaction with guanine: A computational study

    Indian Academy of Sciences (India)

    Bhabesh Chandra Deka; Pradip Kr Bhattacharyya

    2016-04-01

    The present study delves into the reactivity of a few chitosan derivatives (CSDs) and their interaction with guanine in vacuum and in different phases. Increase in the polarity of the solvent lowers reactivity of the chosen derivatives (evaluated by using reactivity descriptors). Interaction between the CSDs and guanine (measured by interaction energy) weakens in solvent media and CSD-guanine interaction is weaker than the interaction between guanine and unmodified chitosan (CS). Chemical stability of CSD-guanine adducts remains similar to that of CS-guanine adduct in both polar and non-polar media. Moreover, CSD-guanine adducts exhibit comparable thermodynamic stability (quantified by free energy of solvation, Gsol) to that of unmodified CS-guanine adduct in non-polar solvent but in polar medium they are immensely destabilized in comparison to CS-guanine adduct. Observed theoretical results are expected to provide guidance for future relevant experimental research on gene delivery by CS derivatives.

  5. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Energy Technology Data Exchange (ETDEWEB)

    Di Paolo, T.; Falardeau, P.

    1987-08-31

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p < 0.001). Competition for (/sup 3/H)-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-(..beta..-..gamma..-imino)triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables.

  6. Identification, Expression, and Characterization of Escherichia coli Guanine Deaminase

    OpenAIRE

    Maynes, Jason T.; Yuan, Richard G.; Snyder, Floyd F.

    2000-01-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a Km of ...

  7. Updating our view of organelle genome nucleotide landscape

    Directory of Open Access Journals (Sweden)

    David Roy Smith

    2012-09-01

    Full Text Available Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT content. Recent studies, however, have uncovered guanine and cytosine (GC-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits.

  8. The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor

    NARCIS (Netherlands)

    Milon, P.; Tischenko, E.V.; Tomsic, J.; Caserta, E.; Folkers, G.E.; La Teana, A.; Rodnina, M.V.; Pon, C.L.; Boelens, R.; Gualerzi, C.O.

    2006-01-01

    Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3′,5′-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low.

  9. Endogenous melatonin and oxidatively damaged guanine in DNA

    DEFF Research Database (Denmark)

    Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan;

    2009-01-01

    BACKGROUND: A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radic...... attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. METHODS: Mother...... overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were...

  10. Effect of base pairing on the electrochemical oxidation of guanine.

    Science.gov (United States)

    Costentin, Cyrille; Hajj, Viviane; Robert, Marc; Savéant, Jean-Michel; Tard, Cédric

    2010-07-28

    The effect of base pairing by cytosine on the electrochemical oxidation of guanine is examined by means of cyclic voltammetry on carefully purified reactants in a solvent, CHCl(3), which strongly favors the formation of an H-bonded pair. The thermodynamics and kinetics of the oxidation reaction are not strongly influenced by the formation of the pair. They are actually similar to those of the reaction in which 2,6-lutidine, an encumbered base that cannot form a pair with guanine, replaces cytosine. The reaction does not entail a concerted proton-electron mechanism, as attested by the absence of H/D isotope effect. It rather involves the rate-determining formation of the cation radical, followed by its deprotonation and dimerization of the resulting neutral radical in competition with its further oxidation.

  11. In vivo formation of N7-guanine DNA adduct by safrole 2',3'-oxide in mice.

    Science.gov (United States)

    Shen, Li-Ching; Chiang, Su-Yin; Lin, Ming-Huan; Chung, Wen-Sheng; Wu, Kuen-Yuh

    2012-09-18

    Safrole, a naturally occurring product derived from spices and herbs, has been shown to be associated with the development of hepatocellular carcinoma in rodents. Safrole 2',3'-oxide (SFO), an electrophilic metabolite of safrole, was shown to react with DNA bases to form detectable DNA adducts in vitro, but not detected in vivo. Therefore, the objective of this study was to investigate the formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SFO-Gua) resulting from the reaction of SFO with the most nucleophilic site of guanine in vitro and in vivo with a newly developed isotope-dilution high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method. N7γ-SFO-Gua and [(15)N(5)]-N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine ([(15)N(5)]-N7γ-SFO-Gua) were first synthesized, purified, and characterized. The HPLC-ESI-MS/MS method was developed to measure N7γ-SFO-Gua in calf thymus DNA treated with 60 μmol of SFO for 72 h and in urine samples of mice treated with a single dose of SFO (30 mg/kg body weight, intraperitoneally). In calf thymus DNA, the level of N7γ-SFO-Gua was 2670 adducts per 10(6)nucleotides. In urine of SFO-treated mice, the levels of N7γ-SFO-Gua were 1.02±0.14 ng/mg creatinine (n=4) on day 1, 0.73±0.68 ng/mg creatinine (n=4) on day 2, and below the limit of quantitation on day 3. These results suggest that SFO can cause in vivo formation of N7γ-SFO-Gua, which may then be rapidly depurinated from the DNA backbone and excreted through urine.

  12. Classifying Coding DNA with Nucleotide Statistics

    Directory of Open Access Journals (Sweden)

    Nicolas Carels

    2009-10-01

    Full Text Available In this report, we compared the success rate of classification of coding sequences (CDS vs. introns by Codon Structure Factor (CSF and by a method that we called Universal Feature Method (UFM. UFM is based on the scoring of purine bias (Rrr and stop codon frequency. We show that the success rate of CDS/intron classification by UFM is higher than by CSF. UFM classifies ORFs as coding or non-coding through a score based on (i the stop codon distribution, (ii the product of purine probabilities in the three positions of nucleotide triplets, (iii the product of Cytosine (C, Guanine (G, and Adenine (A probabilities in the 1st, 2nd, and 3rd positions of triplets, respectively, (iv the probabilities of G in 1st and 2nd position of triplets and (v the distance of their GC3 vs. GC2 levels to the regression line of the universal correlation. More than 80% of CDSs (true positives of Homo sapiens (>250 bp, Drosophila melanogaster (>250 bp and Arabidopsis thaliana (>200 bp are successfully classified with a false positive rate lower or equal to 5%. The method releases coding sequences in their coding strand and coding frame, which allows their automatic translation into protein sequences with 95% confidence. The method is a natural consequence of the compositional bias of nucleotides in coding sequences.

  13. Theoretical Investigation on the Geometries and Properties of Guanine-BX3 (X = F, Cl) Complex

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Geometries and binding energies were predicted at the B3LYP/6-311+G* level for the guanine-BX3 (X = F, Cl) systems and four isomers with no imaginary frequencies have been obtained for both guanine-BF3 and guanine-BCl3, respectively. Single energy calculations using much larger basis sets (6-311+G(2df,p) and aug-cc-pVDZ were carried out as well. It was found that the most stable isomer of guanine-BF3 is BF3 connected to N3 of guanine with the stabilization energy of -19.93 kcal/mol (BSSE corrected), while that of guanine-BCl3 is BCl3 connected to O10 of guanine having stabilization energy of -15.02 kcal/mol at the same level. The analyses for the combining interaction between BX3 and guanine with the atom-in-molecules theory (AIM) and natural bond orbital (NBO) methods have been performed. The results indicated that all the isomers are formed with σ-p type interactions between guanine and BX3, in which pyridine-type nitrogen or carbonyl oxygen or nitrogen atom of amino group offers its lone pair electrons to the empty p orbital of boron atom and the concomitance of charge transfer from guanine to BX3 has occurred. Still, one or two hydrogen bonds exist in some isomers of guanine-BX3 system and contribute to the stability of complex systems. Frequency analysis suggested that the stretching vibration of BX3 undergoes a red shift in complexes. Guanine-BF3 complex is more stable than guanine-BCl3 although the B-Y (Y=N, O) bond distance in the latter is shorter.

  14. Isolation and characterization of human liver guanine deaminase.

    Science.gov (United States)

    Gupta, N K; Glantz, M D

    1985-01-01

    Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine. PMID:3966794

  15. A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

    OpenAIRE

    Hu, Jie; Ma, Ao; Dinner, Aaron R.

    2008-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) repairs damage to the human genome by flipping guanine and thymine bases into its active site for irreversible transfer of alkyl lesions to Cys-145, but how the protein identifies its targets has remained unknown. Understanding molecular recognition in this system, which can serve as a paradigm for the many nucleotide-flipping proteins that regulate genes and repair DNA in all kingdoms of life, is particularly important given that inhibitors are in c...

  16. Repair of triplex-directed DNA alkylation by nucleotide excision repair

    OpenAIRE

    Ziemba, Amy; Derosier, L. Chris; Methvin, Russell; Song, Chun-Yan; Clary, Eric; Kahn, Wendy; Milesi, David; Gorn, Vladimir; Reed, Mike; Ebbinghaus, Scot

    2001-01-01

    Triplex-forming oligonucleotides (TFOs) are being investigated as highly specific DNA binding agents to inhibit the expression of clinically relevant genes. So far, they have been shown to inhibit transcription from the HER-2/neu gene in vitro, whereas their use in vivo has been studied to a limited extent. This study uses a TFO–chlorambucil (chl) conjugate capable of forming site-specific covalent guanine adducts within the HER-2/neu promoter. We demonstrate that nucleotide excision repair (...

  17. The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth

    Directory of Open Access Journals (Sweden)

    Leo Chang

    2015-08-01

    Full Text Available The generation of neuronal morphology requires transport vesicles originating from the Golgi apparatus (GA to deliver specialized components to the axon and dendrites. Drosophila Arfaptin is a membrane-binding protein localized to the GA that is required for the growth of the presynaptic nerve terminal. Here we provide biochemical, cellular and genetic evidence that the small GTPase Arl1 and the guanine-nucleotide exchange factor (GEF Gartenzwerg are required for Arfaptin function at the Golgi during synapse growth. Our data define a new signaling pathway composed of Arfaptin, Arl1, and Garz, required for the generation of normal synapse morphology.

  18. Modification of guanine bases by nucleoside phosphoramidite reagents during the solid phase synthesis of oligonucleotides.

    OpenAIRE

    Pon, R T; Damha, M J; Ogilvie, K K

    1985-01-01

    Nucleoside 3'-phosphoramidite and chlorophosphite reagents have been found to react with the lactam function of guanine. This reaction caused unsatisfactory results when oligodeoxyribonucleotides containing a large number of guanine bases were prepared in an automated solid phase synthesizer. The guanine modification is unstable, and leads to depurination and chain cleavage. This side reaction can be eliminated by protecting the O6-position. A new O6-p-nitrophenylethyldeoxyguanosine phosphora...

  19. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases

    OpenAIRE

    Rogers, Faye A.; Lloyd, Janice A; Tiwari, Meetu Kaushik

    2014-01-01

    Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-r...

  20. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

    Science.gov (United States)

    Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

    2015-01-01

    Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

  1. Self-assembled guanine ribbons as wide-bandgap semiconductors

    Science.gov (United States)

    di Felice, Rosa; Calzolari, Arrigo; Molinari, Elisa; Garbesi, Anna; Rinaldi, Ross; Maruccio, Giuseppe; Cingolani, Roberto

    2002-03-01

    We present a new class of biomolecular wide-bandgap semiconductors, that spontaneously form by the self-assembling of deoxyguanosine molecules (a modified DNA base) in the solid state. Their deposition onto planar metallic nanocircuits allows the fabrication of hybrid nanodiodes and metal/semiconductor/metal devices. By means of first-principle calculations, we describe the structure and the electronic properties of stacked guanine ribbons. We discuss the formation of extended Bloch orbitals, resulting from the superposition of base-localized states, as a function of H-bonding and π-π coupling. The oveall band-like conduction is affected by a dipole-field that spontaneously arise along the ribb n axis. Our theoretical model explains both the basic transport mechanism and the current-voltage characteristics of the devices.

  2. Analysis of difference spectra of protonated DNA: determination of degree of protonation of nitrogen bases and the fractions of disordered nucleotide pairs.

    Science.gov (United States)

    Smol'janinova, T I; Zhidkov, V A; Sokolov, G V

    1982-01-01

    The titration curves of nitrogen bases and fractions of disordered nucleotide pairs are obtained during DNA protonation. It is shown that purine bases are the first sites of the DNA double helix protonation. The cytosine protonation is due to proton-induced conformational transition within GC pairs with the sequence proton transfer from (N-7) of guanine to (N-3) of cytosine. Within DNA with unwound regions the bases are protonated in the following order: cytosine, adenine, guanine. It is shown that GC pairs are the primary centres in which the unwinding of protonated DNAs occurs. PMID:7079177

  3. Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

    Energy Technology Data Exchange (ETDEWEB)

    Caroline Kisker

    2001-01-01

    Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

  4. Enhanced Molecular Recognition between Nucleobases and Guanine-5'-monophosphate-disodium (GMP) by Surfactant Aggregates in Aqueous Solution.

    Science.gov (United States)

    Liu, Zhang; Wang, Dong; Cao, Meiwen; Han, Yuchun; Xu, Hai; Wang, Yilin

    2015-07-15

    Only specific base pairs on DNA can bind with each other through hydrogen bonds, which is called the Watson-Crick (W/C) pairing rule. However, without the constraint of DNA chains, the nucleobases in bulk aqueous solution usually do not follow the W/C pairing rule anymore because of the strong competitive effect of water and the multi-interaction edges of nucleobases. The present work applied surfactant aggregates noncovalently functionalized by nucleotide to enhance the recognition between nucleobases without DNA chains in aqueous solution, and it revealed the effects of their self-assembling ability and morphologies on the recognition. The cationic ammonium monomeric, dimeric, and trimeric surfactants DTAB, 12-3-12, and 12-3-12-3-12 were chosen. The surfactants with guanine-5'-monophosphate-disodium (GMP) form micelles, vesicles, and fingerprint-like and plate-like aggregates bearing the hydrogen-bonding sites of GMP, respectively. The binding parameters of these aggregates with adenine (A), uracil (U), guanine (G), and cytosine(C) indicate that the surfactants can promote W/C recognitions in aqueous solution when they form vesicles (GMP/DTAB) or plate-like aggregates (GMP/12-3-12) with proper molecular packing compactness, which not only provide hydrophobic environments but also shield non-W/C recognition edges. However, the GMP/12-3-12 micelles with loose molecular packing, the GMP/12-3-12 fingerprint-like aggregates where the hydrogen bond sites of GMP are occupied by itself, and the GMP/12-3-12-3-12 vesicles with too strong self-assembling ability cannot promote W/C recognition. This work provides insight into how to design self-assemblies with the performance of enhanced molecule recognition.

  5. Single Nucleotide Polymorphism

    DEFF Research Database (Denmark)

    Børsting, Claus; Pereira, Vania; Andersen, Jeppe Dyrberg;

    2014-01-01

    Single nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations in the genome. They have been studied extensively in the last decade with various purposes in mind. In this chapter, we will discuss the advantages and disadvantages of using SNPs for human identification...

  6. Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA*

    OpenAIRE

    Awai, Takako; Kimura, Satoshi; Tomikawa, Chie; Ochi, Anna; Ihsanawati,; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Ohno, Satoshi; Nishikawa, Kazuya; Yokogawa, Takashi; Suzuki, Tsutomu; Hori, Hiroyuki

    2009-01-01

    Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. I...

  7. Pioneer Axon Navigation Is Controlled by AEX-3, a Guanine Nucleotide Exchange Factor for RAB-3 in Caenorhabditis elegans.

    Science.gov (United States)

    Bhat, Jaffar M; Hutter, Harald

    2016-07-01

    Precise and accurate axon tract formation is an essential aspect of brain development. This is achieved by the migration of early outgrowing axons (pioneers) allowing later outgrowing axons (followers) to extend toward their targets in the embryo. In Caenorhabditis elegans the AVG neuron pioneers the right axon tract of the ventral nerve cord, the major longitudinal axon tract. AVG is essential for the guidance of follower axons and hence organization of the ventral nerve cord. In an enhancer screen for AVG axon guidance defects in a nid-1/Nidogen mutant background, we isolated an allele of aex-3 aex-3 mutant animals show highly penetrant AVG axon navigation defects. These defects are dependent on a mutation in nid-1/Nidogen, a basement membrane component. Our data suggest that AEX-3 activates RAB-3 in the context of AVG axon navigation. aex-3 genetically acts together with known players of vesicular exocytosis: unc-64/Syntaxin, unc-31/CAPS, and ida-1/IA-2. Furthermore our genetic interaction data suggest that AEX-3 and the UNC-6/Netrin receptor UNC-5 act in the same pathway, suggesting AEX-3 might regulate the trafficking and/or insertion of UNC-5 at the growth cone to mediate the proper guidance of the AVG axon. PMID:27116976

  8. Activated RhoA is a positive feedback regulator of the Lbc family of Rho guanine nucleotide exchange factor proteins.

    Science.gov (United States)

    Medina, Frank; Carter, Angela M; Dada, Olugbenga; Gutowski, Stephen; Hadas, Jana; Chen, Zhe; Sternweis, Paul C

    2013-04-19

    The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs.

  9. G-quartet type self-assembly of guanine functionalized single-walled carbon nanotubes

    Science.gov (United States)

    Singh, Prabhpreet; Venkatesh, V.; Nagapradeep, N.; Verma, Sandeep; Bianco, Alberto

    2012-03-01

    The simple strategy of linking guanine to single-walled carbon nanotubes (CNTs) through covalent functionalization permitted generation of the alignment of the nanotubes into lozenges reminiscent of guanine quartets (G-quartets) in the presence of potassium ions as observed by atomic force microscopy.The simple strategy of linking guanine to single-walled carbon nanotubes (CNTs) through covalent functionalization permitted generation of the alignment of the nanotubes into lozenges reminiscent of guanine quartets (G-quartets) in the presence of potassium ions as observed by atomic force microscopy. Electronic supplementary information (ESI) available: Experimental procedures for the synthesis and characterization of the precursors and MWCNT conjugates. See DOI: 10.1039/c2nr11849a

  10. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

    Science.gov (United States)

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-11-01

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

  11. An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation.

    OpenAIRE

    Burke, J F; Sinclair, J H; Sang, J. H.; Ish-Horowicz, D.

    1984-01-01

    We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells...

  12. A novel method for detecting 7-methyl guanine reveals aberrant methylation levels in Huntington disease

    OpenAIRE

    Thomas, Beena; Matson, Samantha; Chopra, Vanita; Sun, Liping; Sharma, Swati; Hersch, Steven; Rosas, H. Diana; Scherzer, Clemens; Ferrante, Robert; Matson, Wayne

    2013-01-01

    Guanine methylation is a ubiquitous process affecting DNA and various RNA species. N-7 guanine methylation (7-MG), though relatively less studied, could have a significant role in normal transcriptional regulation as well as in the onset and development of pathological conditions. The lack of a sensitive method to accurately quantify trace amounts of altered bases like 7-MG, has been a major deterrent in delineating its biological function(s). Here we report the development of methods to dete...

  13. Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

    Directory of Open Access Journals (Sweden)

    Oliva Glaucius

    2007-09-01

    Full Text Available Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT (EC 2.4.2.8 is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

  14. The glycine brace: a component of Rab, Rho, and Ran GTPases associated with hinge regions of guanine- and phosphate-binding loops

    Directory of Open Access Journals (Sweden)

    Neuwald Andrew F

    2009-03-01

    Full Text Available Abstract Background Ras-like GTPases function as on-off switches in intracellular signalling pathways and include the Rab, Rho/Rac, Ran, Ras, Arf, Sar and Gα families. How these families have evolutionarily diverged from each other at the sequence level provides clues to underlying mechanisms associated with their functional specialization. Results Bayesian analysis of divergent patterns within a multiple alignment of Ras-like GTPase sequences identifies a structural component, termed here the glycine brace, as the feature that most distinguishes Rab, Rho/Rac, Ran and (to some degree Ras family GTPases from other Ras-like GTPases. The glycine brace consists of four residues: An aromatic residue that forms a stabilizing CH-π interaction with a conserved glycine at the start of the guanine-binding loop; a second aromatic residue, which is nearly always a tryptophan, that likewise forms stabilizing CH-π and NH-π interactions with a glycine at the start of the phosphate-binding P-loop; and two other residues (typically an aspartate and a serine or threonine that, together with a conserved buried water molecule, form a network of interactions connecting the two aromatic residues. Conclusion It is proposed that the two glycine residues function as hinges and that the glycine brace influences guanine nucleotide binding and release by interacting with these hinges.

  15. Guanosine nucleotide precursor for flavinogenesis of Eremothecium Ashbyii.

    Science.gov (United States)

    Mitsuda, H; Nakajima, K

    1975-01-01

    The purine precursor in the riboflavin biosynthetic pathway in Eremothecium ashbyii was examined using a guanine analogue, 8-azaguanine, with non-growing cell systems. 1. Riboflavin formation in the culture filtrate was determined at 0, 5, 10 and 20 hr after start of the incubation of the non-growing cells in the presence of xanthine or 8-azaguanine (1 mM, respectively). At 20 hr of incubation, the addition of xanthine stimulated riboflavin formation by 36% and the addition of 8-azaguanine inhibited the formation by 57%. 2. Acid soluble nucleotide pools in the cells were followed at 0, 5, 10 and 20 hr of the incubation period in the presence of xanthine or 8-azaguanine by means of anion exchange column chromatography. The result showed that the GTP pool changed markedly despite the fact that the adenosine nucleotide pool was almost constant irrespective of the presence or absence of these purines till 10 hr of incubation. But, the decrease of the former was overcome in part by the addition of flavinogenic xanthine. Furthermore, the total amounts of GTP and guanosine accumulated in cells in the presence of 8-azaguanine reached the maximum already at 5 hr, attaining a level twice as much as the GTP contents of the control. 3. The role of guanosine nucleotide pool in riboflavin formation was further examined using 8-azaguanine. In this experiment the drug was added to the suspension of non-growing cells at 3 hr or 6 hr after the incubation was started and the reaction was continued till the 12th hr. A more clear-cut correlationship between riboflavin formation and guanosine nucleotide pool was oberved by this experiment. The guanosine nucleotide pool (consisting of GMP, GDP and GTP) increased simultaneously with the inhibition of riboflavin formation. Of the guanosine nucleotides pools, the GMP pool increased 2.7 times above normal upon the addition of 8-azaguanine during the incubation for 6 hr and 5.3 fold for 9 hr. While, the GTP pool increased 1.9 fold above

  16. Experimental and first-principles study of guanine adsorption on ZnO clusters.

    Science.gov (United States)

    Chandraboss, V L; Karthikeyan, B; Senthilvelan, S

    2014-11-14

    Theoretical investigation of guanine, DNA base adsorption on the ZnO model clusters, viz., Zn2O2, Zn3O3, Zn4O4 ring (R) and Zn4O4 wurtzite (W) in terms of geometry, binding site, binding energy (EB), energy gap (Eg), electronic and spectral properties were studied by a density functional theory (DFT) method. The guanine adsorption on the ZnO (G-ZnO) clusters is modeled by the B3LYP/LanL2DZ method. The calculated binding energy (EB) and energy gap (Eg) of the guanine molecule are highly dependent on the nature of the cluster size and vary with the size of the clusters. Physisorption proceeded via formation of the NZn bond between guanine and the active Zn(2+) site on ZnO. The HOMO-LUMO energies show that charge transfer occurs in the G-ZnO clusters, from ZnO to guanine to better understand the interaction. The Mulliken charges are computed. The electronic properties of ZnO and G-ZnO clusters were compared with different basis sets (B3LYP/6-31G, B3LYP/6-311G, MP2/6-31G and MP2/LanL2DZ). Experimental information like microscopic and spectroscopic evidence is also included for understanding the guanine-ZnO interactions. The G-ZnO composite was prepared by a precipitation method and characterized by SEM with EDX, FT-IR and FT-RAMAN analysis. The interaction of guanine with ZnO nanoparticles was observed by UV-vis spectroscopy. The experimental results are compared with the DFT results in the light of these new insights. PMID:25266048

  17. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    Science.gov (United States)

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

  18. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes.

    Science.gov (United States)

    Shi, Xianzong; Jarvis, Donald L

    2006-09-15

    Rapid amplification of cDNA ends (RACE) is widely used to determine the 5'- and 3'-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none addresses the difficult problems that arise when trying to isolate the ends of extremely guanine plus cytosine (GC)-rich genes. In this study, we found that we were unable to isolate the correct 5' or 3' end of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first-strand cDNA synthesis at 70 degrees C with Thermo-X reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98 degrees C denaturation steps and Phusion DNA polymerase in the presence of 1M betaine and 5% dimethyl sulfoxide (DMSO). The use of these conditions yielded 5'- and 3'-RACE products that were approximately 80% GC over 213 and 162bp, respectively, and included shorter internal regions of 82 to 89% GC. PMID:16875657

  19. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    Science.gov (United States)

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  20. Examination of the effect of the annealing cation on higher order structures containing guanine or isoguanine repeats

    OpenAIRE

    Pierce, Sarah E.; Wang, Junmei; Jayawickramarajah, Janarthanan; Hamilton, Andrew D.; Brodbelt, Jennifer S.

    2009-01-01

    Isoguanine (2-oxo-6-amino-guanine), a natural but non-standard base, exhibits unique self-association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. ...

  1. Theoretical Study of the Photophysics of 8-Vinylguanine, an Isomorphic Fluorescent Analogue of Guanine.

    Science.gov (United States)

    Kochman, Michał A; Pola, Martina; Miller, R J Dwayne

    2016-08-11

    Paving the way for the application of the algebraic-diagrammatic construction scheme of second-order (ADC(2)) to systems based on the guanine chromophore, we demonstrate the this excited-state electronic structure method provides a realistic description of the photochemistry of 9H-guanine, in close agreement with the benchmark provided by the CASPT2 method. We then proceed to apply the ADC(2) method to the photochemistry of 8-vinylguanine (8vG), a minimally modified analogue of guanine which, unlike the naturally occurring nucleobase, displays intense fluorescence, indicative of a much longer-lived excited electronic state. The emissive electronic state of 8vG is identified as an ππ*-type intramolecular charge transfer (ICT) state, in which a charge of roughly -0.2 e is transferred from the guanine moiety onto the vinyl substituent. The main radiationless deactivation pathway competing with fluorescence is predicted to involve the molecule leaving the minimum on the ICT ππ* state, and reaching a region of the S1 adiabatic state where it resembles the La ππ* state of unmodified 9H-guanine. The topology of the La ππ* region of the S1 state favors subsequent internal conversion at a crossing seam with the ground electronic state. The sensitivity of this process to environment polarity may explain the experimentally observed fluorescence quenching of 8vG upon incorporation in single- and double-stranded DNA. PMID:27427772

  2. Highly sensitive and selective fluorescent assay for guanine based on the Cu2 +/eosin Y system

    Science.gov (United States)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-01

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu2 +/eosin Y. Cu2 + interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu2 +/eosin Y system, guanine reacted with Cu2 + to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L- 1 and a linear range of 3.3-116 nmol L- 1. The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe.

  3. Hypoxanthine-guanine phosophoribosyltransferase (HPRT deficiency: Lesch-Nyhan syndrome

    Directory of Open Access Journals (Sweden)

    Puig Juan G

    2007-12-01

    Full Text Available Abstract Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1/380,000 live births in Canada, and 1/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated with lithiasis and gout. Neurological manifestations include severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit, and self-injurious behaviour. The most severe forms are known as Lesch-Nyhan syndrome (patients are normal at birth and diagnosis can be accomplished when psychomotor delay becomes apparent. Partial HPRT-deficient patients present these symptoms with a different intensity, and in the least severe forms symptoms may be unapparent. Megaloblastic anaemia is also associated with the disease. Inheritance of HPRT deficiency is X-linked recessive, thus males are generally affected and heterozygous female are carriers (usually asymptomatic. Human HPRT is encoded by a single structural gene on the long arm of the X chromosome at Xq26. To date, more than 300 disease-associated mutations in the HPRT1 gene have been identified. The diagnosis is based on clinical and biochemical findings (hyperuricemia and hyperuricosuria associated with psychomotor delay, and enzymatic (HPRT activity determination in haemolysate, intact erythrocytes or fibroblasts and molecular tests. Molecular diagnosis allows faster and more accurate carrier and prenatal diagnosis. Prenatal diagnosis can be performed with amniotic cells obtained by amniocentesis at about 15–18 weeks' gestation, or chorionic villus cells obtained at about 10–12 weeks' gestation. Uric acid overproduction can be managed by allopurinol treatment. Doses must be carefully adjusted to avoid xanthine lithiasis. The

  4. Electronic transitions of guanine tautomers, their stacked dimers, trimers and sodium complexes

    Science.gov (United States)

    Kushwaha, P. S.; Kumar, Anil; Mishra, P. C.

    2004-02-01

    Planar and nonplanar geometries of the keto-N9H and keto-N7H tautomers of the guanine base of DNA as well as the hydrogen bonded complexes of these species with three water molecules each were optimized using the density functional theory at the B3LYP/6-31G ∗ ∗ level. Geometries of the isolated bases were also optimized using the ab initio approach at the MP2/6-31G ∗ ∗ level. The isolated keto-N9H and keto-N7H tautomers as well as their hydrogen bonded complexes with three water molecules each were solvated in bulk water employing the polarized continuum model (PCM) of the self-consistent reaction field theory (SCRF). Stacked dimers and trimers of both the tautomers of guanine were generated by placing the planar forms of the species at a fixed distance of 3.5 Å from the neighboring one and rotating one molecule with respect to the other by 110° for the keto-N9H form and 90° for the keto-N7H form which corresponded to total energy minima at the B3LYP/6-31G ∗ ∗ level. Geometry optimization for the cation of the monomer of guanine was performed at the same level of theory, and its solvation in bulk water was treated using the PCM model of the SCRF theory. The geometries of complexes of the two tautomers of guanine with a Na + ion each were optimized at the B3LYP/6-31G ∗ ∗ level, and the Na + ion is predicted to bind with the keto-N9H tautomer preferentially. While the complex of the keto-N7H form of guanine with three water molecules in gas phase is slightly more stable than the corresponding complex of the keto-N9H form of guanine, the reverse is true in bulk water. Stacking interactions enhance the relative stability of the keto-N9H tautomer over that of the keto-N7H tautomer, suggesting that in bulk solutions, the former would be dominant. Electronic spectra of the isolated tautomers of guanine, those of their complexes with three water molecules each, the (keto-n9h and keto-n7h) cation of guanine, the complexes of the tautomers with a Na + ion

  5. Synthesis of a Pseudodisaccharide α-C-Glycosidically Linked to an 8-Alkylated Guanine

    Directory of Open Access Journals (Sweden)

    Jan Duchek

    2013-04-01

    Full Text Available The synthesis of stable guanofosfocin analogues has attracted considerable attention in the past 15 years. Several guanofosfocin analogues mimicking the three constitutional elements of mannose, ribose, and guanine were designed and synthesized. Interest in ether-linked pseudodisaccharides and 8-alkylated guanines is increasing, due to their potential applications in life science. In this article, a novel guanofosfocin analogue 6, an ether-linked pseudodisaccharide connected α-C-glycosidically to an 8-alkylated guanine, was synthesized in a 10-longest linear step sequence from known diol 13, resulting in an overall yield of 26%. The key steps involve the ring-opening of cyclic sulfate 8 by alkoxide generated from 7 and a reductive cyclization of 4-N-acyl-2,4-diamino-5-nitrosopyrimidine 19 to form compound 6.

  6. Nucleotide discrimination with DNA immobilized in the MspA nanopore.

    Science.gov (United States)

    Manrao, Elizabeth A; Derrington, Ian M; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H

    2011-01-01

    Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices. PMID:21991340

  7. Synthesis and nonenzymatic template-directed polymerization of 2'-amino-2'-deoxythreose nucleotides.

    Science.gov (United States)

    Blain, J Craig; Ricardo, Alonso; Szostak, Jack W

    2014-02-01

    Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2'-amino modified TNA nucleosides (2'-NH2-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2'-NH2-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3'-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C4 RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h(-1) on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM N-(hydroxylethyl)imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h(-1), causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2'-NH2-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems.

  8. Functionalization of single-walled carbon nanotubes with uracil, guanine, thymine and L-alanine

    Science.gov (United States)

    Silambarasan, D.; Iyakutti, K.; Vasu, V.

    2014-06-01

    Experimental investigation of functionalization of oxidized single-walled carbon nanotubes (OSWCNTs) with three nucleic acid bases such as uracil, guanine, thymine and one amino acid, L-alanine is carried out. Initially, the SWCNTs are oxidized by acid treatment. Further, the oxidized SWCNTs are effectively functionalized with aforementioned biological compounds by ultrasonication. The diameter of OSWCNTs has increased after the adsorption of biological compounds. The cumulative Π-Π stacking, hydrogen bond and polar interaction are the key factors to realize the adsorption. The amount of adsorption of each biological compound is estimated. The adsorption of guanine is more among all the four biological compounds.

  9. Spin transport and polarization properties of manganese-doped dual-guanine molecule

    OpenAIRE

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi; Mazidabadi, Hossein; Nourozi, Mohammad

    2014-01-01

    We study the spin transport and polarization properties of manganese-doped dual-guanine molecules connected to graphene leads using non-equilibrium Green's function method. It is shown that a manganese doped dual-guanine molecule is a biological semiconductor and behaves as a prefect spin filter. We show that this semiconductor can behave as a spin switch when the Rashba spin-orbit interaction is considered. In addition, it is shown that, a large conductance is observed due to the Fano-Kondo-...

  10. High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS.

    Science.gov (United States)

    Lorkiewicz, Pawel; Higashi, Richard M; Lane, Andrew N; Fan, Teresa W-M

    2012-01-01

    Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C(18) stationary phase, which enabled global analysis of nucleotides and their (13)C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 minutes of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD(+), NADH, NADP(+), NADPH, cyclic nucleotides, several UDP-hexoses, and all their (13)C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-(13)C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, (13)C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their (13)C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.

  11. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  12. Ligand-induced folding of the guanine-sensing riboswitch is controlled by a combined predetermined induced fit mechanism

    NARCIS (Netherlands)

    Ottink, O.M.; Rampersad, S.M.; Tessari, M.; Zaman, G.J.; Heus, H.A.; Wijmenga, S.S.

    2007-01-01

    All known guanine-sensing riboswitches regulate gene expression by specifically binding to guanine (G) or related analogs with high affinity to switch off transcription. The aptamers of this class of riboswitches are characterized by three helices (P1-P3), surrounding a central core of phylogenetica

  13. Theoretical and Experimental Study of Valence-Shell Ionization Spectra of Guanine

    Science.gov (United States)

    Zaytseva, Irina L.; Trofimov, Alexander B.; Schirmer, Jochen; Plekan, Oksana; Feyer, Vitaliy; Richter, Robert; Coreno, Marcello; Prince, Kevin C.

    2009-10-01

    The full valence-shell ionization spectra of the four most stable guanine tautomers were studied theoretically. The third-order algebraic-diagrammatic construction (ADC(3)) method for the one-particle Green's function was used to calculate the energies and relative intensities of the vertical ionization transitions. For low-lying transitions, the influence of planar and nonplanar guanine configurations on the ionization energies, as well as the convergence of the results with respect to basis set was studied at the level of the outer-valence Green's function (OVGF) approximation scheme. The results of the calculations were used to interpret recent synchrotron radiation valence-shell photoionization spectra of guanine in the gas phase under thermal equilibrium conditions. The photoelectron spectrum was modeled by summing individual tautomer spectra weighted by Boltzmann population ratios (BPR) of tautomers from our previous high-level ab initio thermochemical calculations. The theoretical spectra are in good agreement with the experimental results, providing assignments of most observed structures and offering insight into tautomerism of guanine in the gas phase. The first six molecular orbitals give rise to single-hole states with a binding energy of about 7-12 eV. At higher binding energy the spectral features are mainly due to satellite states.

  14. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases.

    Science.gov (United States)

    Rogers, Faye A; Lloyd, Janice A; Tiwari, Meetu Kaushik

    2014-01-01

    Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis. PMID:25483840

  15. Partial hypoxanthine-guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion

    NARCIS (Netherlands)

    van Dael, C. M. L.; Pierik, L. J. W. M.; Reijngoud, D. J.; Niezen-Koning, K. E.; van Diggelen, O. P.; van Spronsen, F. J.

    2007-01-01

    Partial hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley-Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and

  16. Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content

    NARCIS (Netherlands)

    Vos, Willem M. de; Kleerebezem, Michiel; Kuipers, Oscar P.

    1997-01-01

    Recent years have seen an increase in the development of gene expression systems for industrial Gram-positive bacteria with low guanine and cytosine content that belong to the genera Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus. In particular, considerable adva

  17. Separation of Guanine and Hypoxanthine with Some Ionic Liquids in RP-HPLC

    Directory of Open Access Journals (Sweden)

    J. Zheng

    2006-01-01

    Full Text Available In this paper, guanine and hypoxanthine were separated with four different ionic liquids as additives for the mobile phase using reversed phase high performance liquid chromatography (RP-HPLC. The ionic liquids, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4], 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF4], 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS] and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS] were used. Guanine and hypoxanthine couldn’t be separated with many different kinds of unadjusted mobile phase, such as aqueous-methanol, aqueous-acetonitrile, etc. In this reason, present study introduced the ionic liquid for separation of guanine and hypoxanthine as an eluent modifier. And the effects of length of alkyl on the imidazolium ring and its counterion, the concentrations of ionic liquid on the retention factor and effect of pH of mobile phase on retention factor of solutes were investigated also. As a result, guanine and hypoxanthine were separated with the mobile phase including ionic liquid and the excellent separations of these sorbats were achieved using 2.0 mM Lˉ1 [OMIm][MS] as the eluent modifier.

  18. Guanine-containing copper(II) complexes: synthesis, X-ray structures and magnetic properties.

    Science.gov (United States)

    Mastropietro, Teresa F; Armentano, Donatella; Grisolia, Ettore; Zanchini, Claudia; Lloret, Francesc; Julve, Miguel; De Munno, Giovanni

    2008-01-28

    Three new compounds of formula {[Cu(gua)(H(2)O)(3)](BF(4))(SiF(6))(1/2)}(n) (1), {[Cu(gua)(H(2)O)(3)](CF(3)SO(3))(2).H(2)O}(n) (2) and [Cu(gua)(2)(H(2)O)(HCOO)]ClO(4).H(2)O.1/2HCOOH] (3) [gua = 2-amino-1H-purin-6(9H)-one] showing the unprecedented coordination of neutral guanine, have been synthesised and structurally characterized. The structures of the compounds 1 and 2 contain uniform copper(II) chains of formula [Cu(gua)(H(2)O)(3)](n)(2n+), where the copper atoms are bridged by guanine ligands coordinated via N(3) and N(7). The electroneutrality is achieved by uncoordinated tetrafluoroborate and hexafluorosilicate (1) and triflate (2). Each copper atom in 1 and 2 is five-coordinated in a distorted square pyramidal environment: two water molecules in trans positions and the N(3) and N(7a) nitrogen atoms of two guanine ligands build the basal plane whereas a water molecule fills the axial position. The values of the copper-copper separation across the bridging guanine ligand are 7.183(1) (1) and 7.123(1) A (2). is an ionic salt whose structure is made up of mononuclear [Cu(gua)(2)(H(2)O)(HCOO)](+) cations and perchlorate anions plus water and formic acid as crystallization molecules. The two guanine ligands in the cation are coordinated to the copper centre through the N(9) atom. The copper atom in 3 is four-coordinated with two monodentate guanine molecules in the trans position, a water molecule and a monodenate formate ligand building a quasi square planar surrounding. Magnetic susceptibility measurements for 1 and 2 in the temperature range 1.9-300 K show the occurrence of significant intrachain antiferromagnetic interactions between the copper(ii) ions across the guanine bridge [J = -9.6(1) (1) and -10.3(1) cm(-1) (2) with H = -J summation operator(i)S(i).S(i+1)].

  19. Hydrogen-bonded proton transfer in the protonated guanine-cytosine (GC+H)+ base pair.

    Science.gov (United States)

    Lin, Yuexia; Wang, Hongyan; Gao, Simin; Schaefer, Henry F

    2011-10-13

    The single proton transfer at the different sites of the Watson-Crick (WC) guanine-cytosine (GC) DNA base pair are studied here using density functional methods. The conventional protonated structures, transition state (TS) and proton-transferred product (PT) structures of every relevant species are optimized. Each transition state and proton-transferred product structure has been compared with the corresponding conventional protonated structure to demonstrate the process of proton transfer and the change of geometrical structures. The relative energies of the protonated tautomers and the proton-transfer energy profiles in gas and solvent are analyzed. The proton-transferred product structure G(+H(+))-H(+)C(N3)(-H(+))(PT) has the lowest relative energy for which only two hydrogen bonds exist. Almost all 14 isomers of the protonated GC base pair involve hydrogen-bonded proton transfer following the three pathways, with the exception of structure G-H(+)C(O2). When the positive charge is primarily "located" on the guanine moiety (H(+)G-C, G-H(+)C(C4), and G-H(+)C(C6)), the H(1) proton transfers from the N(1) site of guanine to the N(3) site of cytosine. The structures G-H(+)C(C5) and G-H(+)C(C4) involve H(4a) proton transfer from the N(4) of cytosine to the O(6) site of guanine. H(2a) proton transfer from the N(2) site of guanine to the O(2) site of cytosine is found only for the structure G-H(+)C(C4). The structures to which a proton is added on the six-centered sites adjoining the hydrogen bonds are more prone to proton transfer in the gas phase, whereas a proton added on the minor groove and the sites adjoining the hydrogen bonds is favorable to the proton transfer in energy in the aqueous phase.

  20. MD Calculation of Ground-State Energy of the Nucleotide Doublets, Their Eigenmode Frequencies and Geometry Optimization

    CERN Document Server

    Kretov, D A

    2004-01-01

    Based on the computer molecular dynamics (MD) modeling methods we have calculated the ground-state energy and eigenvalue frequencies of the adenine-thymine and guanine-cytosine nucleotide doublets and performed their geometry optimization. We have found the eigenmodes in the infrared and radio-frequency ranges. The possibility of application of the simulation results in detection of bio-structures through the spectral analysis is considered. In the quantum chemistry MD calculations we employed the \\textit{Gaussian98} code and MOLDEN package.

  1. Nucleotide sequence analysis of the hypervariable region III of mitochondrial DNA in Thais.

    Science.gov (United States)

    Thongngam, Punlop; Leewattanapasuk, Worraanong; Bhoopat, Tanin; Sangthong, Padchanee

    2016-07-01

    This study analyzed the nucleotide sequences of the hypervariable region III (HVRIII) of mitochondrial DNA in Thai individuals. Buccal swab samples were randomly obtained from 100 healthy, unrelated, adult (18-60 years old), volunteer donors living in Thailand. Eighteen different haplotypes were found, of which 11 haplotypes were unique. The most frequent haplotypes observed were 522D-523D. Nucleotide transition from Thymine (T) to Cytosine (C) at position 489 (43%) was the most frequent substitution. Nucleotide transversions were also observed at position 433 (Adenine (A) to C, 1%) and position 499 (Guanine (G) to C, 1%). Fifty-three samples presented nucleotide insertion and deletion of C and A (CA) at position 514-523. Insertion of 1AC (3%) and 2AC (2%) were observed. Deletion of 1CA (53%) and 2CA (2%) at position 514-523 were revealed. The deletion of T at position 459 was observed. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.7770, 0.2308, and 0.7692, respectively. PMID:27107562

  2. Rapid magnetic wiper featuring biogenic guanine particles: Magnetic non-contact switching of opto-fluidic mirrors featuring biogenic guanine crystals

    Science.gov (United States)

    Iwasaka, M.; Mizukawa, Y.; Miyashita, Y.

    2014-01-01

    In the present study, we prepared a diamagnetic fluid with magnetically controlled visual transparency. Light scattering control by the magnetic orientation of organic and diamagnetic microcrystals was applied for developing a functional diamagnetic fluid, by controlling its turbidity and transparency. The light scattering fluid was prepared by guanine crystal particles suspension (GPS), which were collected from the scales of goldfish, in an aqueous solution. It is revealed that GPS can control light scattering with magnetic fields. The method we developed can be utilized for controlling the visibility of light-reflective objects by using magnetic fields on a millitesla scale.

  3. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  4. Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

    NARCIS (Netherlands)

    LaRowe, D.E.; Regnier, P.

    2008-01-01

    The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditi

  5. Activation of transfer RNA-guanine ribosyltransferase by protein kinase C.

    OpenAIRE

    Morris, R C; Brooks, B. J.; Eriotou, P; Kelly, D F; Sagar, S.; Hart, K L; Elliott, M.S.

    1995-01-01

    Transfer RNA-guanine ribosyltransferase (TGRase) irreversibly incorporates queuine into the first position in the anticodon of four tRNA isoacceptors. Rat brain protein kinase C (PKC) was shown to stimulate rat liver TGRase activity. TGRase preparations derived from rat liver have been observed to decrease in activity over time in storage at -20 or -70 degrees C. Contamination of the samples by phosphatases was indicated by a p-nitrophenylphosphate conversion test. The addition of micromolar ...

  6. Structure–function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase

    OpenAIRE

    Zheng, Sushuang; Shuman, Stewart

    2008-01-01

    The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure–function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal m...

  7. N‐methylmesoporphyrin IX fluorescence as a reporter of strand orientation in guanine quadruplexes

    OpenAIRE

    Sabharwal, Navin C.; Savikhin, Victoria; Turek‐Herman, Joshua R.; Nicoludis, John M.; Szalai, Veronika A.; Yatsunyk, Liliya A.

    2014-01-01

    Guanine quadruplexes (GQ) are four‐stranded DNA structures formed by guanine‐rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs in vivo has proven difficult, in part because of their structural diversity. The development of GQ‐selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N‐methylmesoporphyrin IX (NMM) interacts ...

  8. Structure-function Relationships in Human Hypoxanthine-guanine Phosphoribosyltransferase (HGPRT) by Random Mutagenesis

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Introduction Hypoxanthine-guanine phosphoribosyltransferase ( HGPRT, EC 2.4.2.8 ) is a key enzyme of the purine salvage pathway, which allows recycling of purine bases into DNA and RNA. It is widely distributed in nature and has been studied both in prokaryotes and eukaryotes. In humans, a complete lack of HGPRT activity causes the Lesch-Nyhan syndrome, which is characterized by hyperuricaemia and neural disorders, including mental retardation and compulsive self-mutilation behavior[1].

  9. O 6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts

    OpenAIRE

    Clemons, M.; Kelly, J.; Watson, A.J.; Howell, A.; McElhinney, R S; McMurry, T B H; Margison, G P

    2005-01-01

    Tumour resistance to chemotherapy involving methylating agents such as DTIC (dacarbazine) and temozolomide is linked to expression of the DNA repair protein O 6-alkylguanine-DNA alkyltransferase (MGMT). There is considerable interest in improving the efficacy of such O 6-alkylating chemotherapy by the prior inactivation of MGMT. We have examined the effect of the modified guanine base, O 6-(4-bromothenyl)guanine (PaTrin-2, Patrin™, Lomeguatrib) on MGMT activity and cell or xenograft tumour gr...

  10. N7-(carboxymethyl)guanine-Lithium Crystalline Complex: A Bioinspired Solid Electrolyte

    Science.gov (United States)

    Dutta, Dipak; Nagapradeep, N.; Zhu, Haijin; Forsyth, Maria; Verma, Sandeep; Bhattacharyya, Aninda J.

    2016-04-01

    Electrochemical device with components having direct significance to biological life processes is a potent futuristic strategy for the realization of all-round green and sustainable development. We present here synthesis design, structural analysis and ion transport of a novel solid organic electrolyte (G7Li), a compound reminiscent of ion channels, derived from regioisomeric N7-guanine-carboxylate conjugate and Li-ions. G7Li, with it’s in-built supply of Li+-ions, exhibited remarkably high lithium-ion transference number (= 0.75) and tunable room temperature ionic conductivity spanning three decades (≈10‑7 to 10‑3 Ω‑1 cm‑1) as a function of moisture content. The ionic conductivity show a distinct reversible transition around 80–100 °C, from a dual Li+ and H+ (100 °C). Systematic studies reveal a transition from water-assisted Li-ion transport to Li hopping-like mechanism involving guanine-Li coordination. While as-synthesized G7Li has potential in humidity sensors, the anhydrous G7Li is attractive for rechargeable batteries.

  11. The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts.

    Science.gov (United States)

    Wyss, Laura A; Nilforoushan, Arman; Williams, David M; Marx, Andreas; Sturla, Shana J

    2016-08-19

    Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide BENZI: opposite adducts, with up to 150-fold higher catalytic efficiency for O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies. PMID:27378785

  12. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  13. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

    Science.gov (United States)

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi; Fujii, Kentaro; Yokoya, Akinari; Fukuda, Yoshihiro; Saitoh, Yuji

    2014-08-01

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5'-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5'-monophosphate, and adenosine 5'-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  14. Expression of microRNAs in Horse Plasma and Their Characteristic Nucleotide Composition

    Science.gov (United States)

    Lee, Seungwoo; Hwang, Seungwoo; Yu, Hee Jeong; Oh, Dayoung; Choi, Yu Jung; Kim, Myung-Chul; Kim, Yongbaek; Ryu, Doug-Young

    2016-01-01

    MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition. PMID:26731407

  15. Expression of microRNAs in Horse Plasma and Their Characteristic Nucleotide Composition.

    Directory of Open Access Journals (Sweden)

    Seungwoo Lee

    Full Text Available MicroRNAs (miRNAs in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition.

  16. Applications of adenine nucleotide measurements in oceanography

    Science.gov (United States)

    Holm-Hansen, O.; Hodson, R.; Azam, F.

    1975-01-01

    The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.

  17. Fragmentation of the adenine and guanine molecules induced by electron collisions

    Energy Technology Data Exchange (ETDEWEB)

    Minaev, B. F., E-mail: bfmin@rambler.ru, E-mail: boris@theochem.kth.se [Bohdan Khmelnitsky National University, 18031 Cherkasy (Ukraine); Tomsk State University, 634050 Tomsk (Russian Federation); Shafranyosh, M. I.; Svida, Yu. Yu; Sukhoviya, M. I.; Shafranyosh, I. I. [Uzhgorod National University, 88000 Uzhgorod (Ukraine); Baryshnikov, G. V.; Minaeva, V. A. [Bohdan Khmelnitsky National University, 18031 Cherkasy (Ukraine)

    2014-05-07

    Secondary electron emission is the most important stage in the mechanism of radiation damage to DNA biopolymers induced by primary ionizing radiation. These secondary electrons ejected by the primary electron impacts can produce further ionizations, initiating an avalanche effect, leading to genome damage through the energy transfer from the primary objects to sensitive biomolecular targets, such as nitrogenous bases, saccharides, and other DNA and peptide components. In this work, the formation of positive and negative ions of purine bases of nucleic acids (adenine and guanine molecules) under the impact of slow electrons (from 0.1 till 200 eV) is studied by the crossed electron and molecular beams technique. The method used makes it possible to measure the molecular beam intensity and determine the total cross-sections for the formation of positive and negative ions of the studied molecules, their energy dependences, and absolute values. It is found that the maximum cross section for formation of the adenine and guanine positive ions is reached at about 90 eV energy of the electron beam and their absolute values are equal to 2.8 × 10{sup −15} and 3.2 × 10{sup −15} cm{sup 2}, respectively. The total cross section for formation of the negative ions is 6.1 × 10{sup −18} and 7.6 × 10{sup −18} cm{sup 2} at the energy of 1.1 eV for adenine and guanine, respectively. The absolute cross-section values for the molecular ions are measured and the cross-sections of dissociative ionization are determined. Quantum chemical calculations are performed for the studied molecules, ions and fragments for interpretation of the crossed beams experiments.

  18. Ab Initio Study of the Electron Transfer in an Ionized Stacked Complex of Guanines

    Science.gov (United States)

    Cauët, Emilie; Liévin, Jacques

    2009-08-01

    The charge transfer process in an ionized stacking of two consecutive guanines (G5'G3')+ has been studied by means of state-averaged CASSCF/MRCI and RASSCF/RASPT2 calculations. The ground and two first excited states of the radical cation have been characterized, and the topology of the corresponding potential energy surfaces (PESs) has been studied as a function of all intermolecular geometrical parameters. The results demonstrate that the charge transfer process in (G5'G3')+ is governed by the avoiding crossing between the ground and first excited states of the complex. Relative translation motions of both guanines in their molecular planes are shown to lead to the charge migration between G5' and G3'. Five stationary points (three minima and two saddle points) have been characterized along the reaction path describing the passage of the positive charge from G5' to G3'. The global minimum on the PES is found to correspond to the charge configuration G5'+G3'. The existence of an intermediate minimum along the reaction path has been established, characterizing a structure where the positive charge is equally distributed between the two guanines. The calculated energy profile allowed us to determine the height of the potential energy barrier (7.33 kcal/mol) and to evaluate the electronic coupling at a geometry close to the avoiding crossing (3.6 kcal/mol). Test calculations showed that the topology of the ground state PES of the complex GG+ is qualitatively conserved upon optimization of the intramolecular geometrical parameters of the stationary points.

  19. Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro

    Indian Academy of Sciences (India)

    Swathi Kota; Hari S Misra

    2015-12-01

    Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase lb of D. radiodurans (DraTopolB) could change the electrophoretic mobility of fast migrating intramolecular rec-G4 DNA into the slow migrating species. DraTopolB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular rec-G4 DNA structures stabilized by K+. On the contrary, when DraTopolB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopolB. This implies that DraTopolB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopolB activity on intramolecular G4 DNA structures. These results suggested that DraTopolB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

  20. Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro.

    Science.gov (United States)

    Kota, Swathi; Misra, Hari S

    2015-12-01

    Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

  1. Pt(II) and Pd(II) complexes of isocytosine and guanine ligands

    OpenAIRE

    Gupta, Deepali

    2005-01-01

    The central idea behind the present project was the synthesis of the cationic di- and trinucleotide analogs with a positively charged Pt2+ backbone. The nucleobases can attach to the metal centers either through a) N1 (unsubstituted isocytosine base), via b) N9 (unsubstituted guanine base), c) N7 (9-EtGH) or d) N9 (7-MeGH) These cationic oligonucleotides are expected to be kinetically robust. Moreover, the nucleobases attached to the metal centers are forced in a more or les...

  2. N7-(carboxymethyl)guanine-Lithium Crystalline Complex: A Bioinspired Solid Electrolyte

    OpenAIRE

    Dipak Dutta; N. Nagapradeep; Haijin Zhu; Maria Forsyth; Sandeep Verma; Aninda J. Bhattacharyya

    2016-01-01

    Electrochemical device with components having direct significance to biological life processes is a potent futuristic strategy for the realization of all-round green and sustainable development. We present here synthesis design, structural analysis and ion transport of a novel solid organic electrolyte (G7Li), a compound reminiscent of ion channels, derived from regioisomeric N7-guanine-carboxylate conjugate and Li-ions. G7Li, with it’s in-built supply of Li+-ions, exhibited remarkably high l...

  3. Solvent effect on the anharmonic vibrational frequencies in guanine-cytosine base pair

    Science.gov (United States)

    Bende, A.; Muntean, C. M.

    2012-02-01

    We present an ab initio study of the vibrational properties of cytosine and guanine in the Watson-Crick and Hoogsteen base pair configurations. The results are obtained by considering the DFT method together with the Polarizable Continuum Model (PCM) using PBE and B3PW91 exchange-correlation functionals and triple-ζ valence basis set. We investigate the importance of anharmonic corrections for the vibrational modes taking into account the solvent effect of the water environment. In particular, the unusual anharmonic effect of the H+ vibration in the case of the Hoogsteen base pair configuration is discussed.

  4. Direct Experimental Observation of the Low Ionization Potentials of Guanine in Free Oligonucleotides by Using Photoelectron Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xin; Wang, Xue B.; Vorpagel, Erich R.; Wang, Lai S.

    2004-12-21

    Photodetachment photoelectron spectroscopy is used to probe the electronic structure of mono-, di-, and trinucleotide anions in the gas phase. A weak and well defined threshold band was observed in the photoelectron spectrum of 2' -deoxyguanosine 5' -mono-phosphate at a much lower ionization energy than the other three mononucleotides. Density function theory calculations revealed that this unique spectral feature is caused by electron-detachment from a ? orbital of the guanine base on 2? ?deoxyguanosine 5? ?monophosphate, whereas the lowest ionization channel for the other three mononucleotides takes place from the phosphate group. This low-energy feature was shown to be a ?fingerprint? in all the spectra of dinucleotides and trinucleotides that contain the guanine base. The current experiment provides direct spectroscopic evidence that the guanine base is the site with the lowest ionization potential in oligonucleotides and DNA and is consistent with the fact that guanine is most susceptible to oxidation to give the guanine cation in DNA damage.

  5. Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

    Science.gov (United States)

    Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

    2015-05-01

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

  6. The research progress on function of Ras guanyl nucleotide releasing protein family%Ras 鸟苷酸释放蛋白家族的功能研究新进展

    Institute of Scientific and Technical Information of China (English)

    杨柳; 陶娟; 涂亚庭

    2015-01-01

    Ras鸟苷酸释放蛋白家族(Ras guanyl nucleotide releasing proteins ,RasGRPs)是鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factors ,GEFs)中的一员,由4种类型的蛋白质组成。 RasGRPs能通过交换鸟嘌呤核苷酸将Ras蛋白从无活性的GDP形式变成有活性的GTP形式,从而活化Ras。4种不同类型的RasGRP蛋白拥有共同的分子结构。其结构包括一个由Ras 交换中心和CDC25区域组成的催化中心,同时还拥有一对非典型的EF臂及C1端。异常表达的不同RasGRP蛋白在不同的疾病发病过程中发挥作用。%Ras guanyl nucleotide releasing proteins ( RasGRPs) were one of guanine nucleotide exchange factors .They consist of four types of proteins .RasGRPs can activate Ras from GDP form to GTP form through the exchange of guanine nucleotide .Four differ-ent types of RasGRP proteins share a common molecular structure .The structure consists of a catalytic center including Ras exchange centers and CDC25 region.They also have a pair of atypical EF arms and C 1 side.The abnormal expressions of different RasGRP pro-teins play a role in different diseases .

  7. Reactions of the OOH radical with guanine: Mechanisms of formation of 8-oxoguanine and other products

    Science.gov (United States)

    Kumar, Nagendra; Shukla, P. K.; Mishra, P. C.

    2010-09-01

    The mutagenic product 8-oxoguanine (8-oxoGua) is formed due to intermediacy of peroxyl (OOR) radicals in lipid peroxidation and protein oxidation-induced DNA damage. The mechanisms of these reactions are not yet understood properly. Therefore, in the present study, the mechanisms of formation of 8-oxoGua and other related products due to the reaction of the guanine base of DNA with the hydroperoxyl radical (OOH) were investigated theoretically employing the B3LYP and BHandHLYP hybrid functionals of density functional theory and the polarizable continuum model for solvation. It is found that the reaction of the OOH radical with guanine can occur following seven different mechanisms leading to the formation of various products including 8-oxoGua, its radicals, 5-hydroxy-8-oxoguanine and CO 2. The mechanism that yields 8-oxoGua as an intermediate and 5-hydroxy-8-oxoGua as the final product was found to be energetically most favorable.

  8. Oxidatively generated damage to the guanine moiety of DNA: Mechanistic aspects and formation in cells

    International Nuclear Information System (INIS)

    Nuclear DNA and other molecules in living systems are continuously exposed to endogenously generated oxygen species. Such species range from the unreactive superoxide radical (O2.-)-the precursor of hydrogen peroxide (H2O2)-to the highly reactive hydroxyl radical (OH.). Exogenous chemical and physical agents, such as ionizing radiation and the UVA component of solar light, can also oxidatively damage both the bases and the 2-deoxyribose moieties of cellular DNA. Over the last two decades, researchers have made major progress in understanding the oxidation degradation pathways of DNA that are most likely to occur from either oxidative metabolism or exposure to various exogenous agents. In the first part of this Account, we describe the mechanistic features of one-electron oxidation reactions of the guanine base in isolated DNA and related model compounds. These reactions illustrate the complexity of the various degradation pathways involved. Then, we briefly survey the analytical methods that can detect low amounts of oxidized bases and nucleosides in cells as they are formed. Recent data on the formation of oxidized guanine residues in cellular DNA following exposure to UVA light, ionizing radiation, and high-intensity UV pulses are also provided. We discuss these chemical reactions in the context of (OH.) radical, singlet oxygen, and two-quantum photoionization processes. (authors)

  9. Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

    OpenAIRE

    Bushman, J L; Asuru, A I; Matts, R L; Hinnebusch, A G

    1993-01-01

    Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent o...

  10. SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses

    OpenAIRE

    Fukaya, M; Kamata, A.; Hara, Y; Tamaki, H.; Katsumata, O.; Ito, N.; Takeda, S.; Hata, Y; Suzuki, T.; Watanabe, M.(Niigata University, 950-2181, Niigata, Japan); Harvey, R J; Sakagami, H.

    2011-01-01

    SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelec...

  11. SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses.

    Science.gov (United States)

    Fukaya, Masahiro; Kamata, Akifumi; Hara, Yoshinobu; Tamaki, Hideaki; Katsumata, Osamu; Ito, Naoki; Takeda, Shin'ichi; Hata, Yutaka; Suzuki, Tatsuo; Watanabe, Masahiko; Harvey, Robert J; Sakagami, Hiroyuki

    2011-03-01

    SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both β-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM. PMID:21198641

  12. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

  13. Antibody of predetermined specificity to a carboxy-terminal region of H-ras gene products inhibits their guanine nucleotide-binding function.

    OpenAIRE

    Srivastava, S. K.; Lacal, J C; Reynolds, S.H.; Aaronson, S A

    1985-01-01

    The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecul...

  14. A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1.

    NARCIS (Netherlands)

    Wessels, E.; Duijsings, D.M.; Niu, T.K.; Neumann, S.; Oorschot, V.M.J.; Lange, F. de; Lanke, K.H.W.; Klumperman, J.; Henke, A.; Jackson, C.L.; Melchers, W.J.G.; Kuppeveld, F.J.M. van

    2006-01-01

    Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in in

  15. Phytoestrogens regulate mRNA and protein levels of guanine nucleotide-binding protein, beta-1 subunit (GNB1) in MCF-7 cells.

    Science.gov (United States)

    Naragoni, Srivatcha; Sankella, Shireesha; Harris, Kinesha; Gray, Wesley G

    2009-06-01

    Phytoestrogens (PEs) are non-steroidal ligands, which regulate the expression of number of estrogen receptor-dependent genes responsible for a variety of biological processes. Deciphering the molecular mechanism of action of these compounds is of great importance because it would increase our understanding of the role(s) these bioactive chemicals play in prevention and treatment of estrogen-based diseases. In this study, we applied suppression subtractive hybridization (SSH) to identify genes that are regulated by PEs through either the classic nuclear-based estrogen receptor or membrane-based estrogen receptor pathways. SSH, using mRNA from genistein (GE) treated MCF-7 cells as testers, resulted in a significant increase in GNB1 mRNA expression levels as compared with 10 nM 17beta estradiol or the no treatment control. GNB1 mRNA expression was up regulated two- to fivefold following exposure to 100.0 nM GE. Similarly, GNB1 protein expression was up regulated 12- to 14-fold. GE regulation of GNB1 was estrogen receptor-dependent, in the presence of the anti-estrogen ICI-182,780, both GNB1 mRNA and protein expression were inhibited. Analysis of the GNB1 promoter using ChIP assay showed a PE-dependent association of estrogen receptor alpha (ERalpha) and beta (ERbeta) to the GNB1 promoter. This association was specific for ERalpha since association was not observed when the cells were co-incubated with GE and the ERalpha antagonist, ICI. Our data demonstrate that the levels of G-protein, beta-1 subunit are regulated by PEs through an estrogen receptor pathway and further suggest that PEs may control the ratio of alpha-subunit to beta/gamma-subunits of the G-protein complex in cells. J. Cell. Physiol. 219: 584-594, 2009. (c) 2009 Wiley-Liss, Inc. PMID:19170076

  16. Phytoestrogens Regulate mRNA and Protein Levels of Guanine Nucleotide-Binding Protein, Beta-1 Subunit (GNB1) in MCF-7 Cells

    OpenAIRE

    Naragoni, Srivatcha; Sankella, Shireesha; Harris, Kinesha; Gray, Wesley G.

    2009-01-01

    Phytoestrogens (PEs) are non-steroidal ligands which regulate the expression of number of estrogen receptor-dependent genes responsible for a variety of biological processes. Deciphering the molecular mechanism of action of these compounds is of great importance because it would increase our understanding of the role(s) these bioactive chemicals play in prevention and treatment of estrogen-based diseases. In this study, we applied suppression subtractive hybridization (SSH) to identify genes ...

  17. Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

    Directory of Open Access Journals (Sweden)

    Roberto Pérez-Torrado

    Full Text Available In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

  18. Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

    Science.gov (United States)

    Freitas-Mesquita, Anita Leocadio; Gomes, Marta T; Vieira, Danielle P; Paes-Vieira, Lisvane; Nascimento, Michelle T C; Lopes, Angela H C S; Meyer-Fernandes, José Roberto

    2016-10-01

    The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target. PMID:27531705

  19. Hydroxyl radical (OH•) reaction with guanine in an aqueous environment: a DFT study.

    Science.gov (United States)

    Kumar, Anil; Pottiboyina, Venkata; Sevilla, Michael D

    2011-12-22

    The reaction of hydroxyl radical (OH(•)) with DNA accounts for about half of radiation-induced DNA damage in living systems. Previous literature reports point out that the reaction of OH(•) with DNA proceeds mainly through the addition of OH(•) to the C═C bonds of the DNA bases. However, recently it has been reported that the principal reaction of OH(•) with dGuo (deoxyguanosine) is the direct hydrogen atom abstraction from its exocyclic amine group rather than addition of OH(•) to the C═C bonds. In the present work, these two reaction pathways of OH(•) attack on guanine (G) in the presence of water molecules (aqueous environment) are investigated using the density functional theory (DFT) B3LYP method with 6-31G* and 6-31++G** basis sets. The calculations show that the initial addition of the OH(•) at C(4)═C(5) double bond of guanine is barrier free and the adduct radical (G-OH(•)) has only a small activation barrier of ca. 1-6 kcal/mol leading to the formation of a metastable ion-pair intermediate (G(•+)---OH(-)). The formation of ion-pair is a result of the highly oxidizing nature of the OH(•) in aqueous media. The resulting ion-pair (G(•+)---OH(-)) deprotonates to form H(2)O and neutral G radicals favoring G(N(1)-H)(•) with an activation barrier of ca. 5 kcal/mol. The overall process from the G(C(4))-OH(•) (adduct) to G(N(1)-H)(•) and water is found to be exothermic in nature by more than 13 kcal/mol. (G-OH(•)), (G(•+)---OH(-)), and G(N(1)-H)(•) were further characterized by the CAM-B3LYP calculations of their UV-vis spectra and good agreement between theory and experiment is achieved. Our calculations for the direct hydrogen abstraction pathway from N(1) and N(2) sites of guanine by the OH(•) show that this is also a competitive route to produce G(N(2)-H)(•), G(N(1)-H)(•) and H(2)O.

  20. The guanine cation radical: investigation of deprotonation states by ESR and DFT.

    Science.gov (United States)

    Adhikary, Amitava; Kumar, Anil; Becker, David; Sevilla, Michael D

    2006-11-30

    This work reports ESR studies that identify the favored site of deprotonation of the guanine cation radical (G*+) in an aqueous medium at 77 K. Using ESR and UV-visible spectroscopy, one-electron oxidized guanine is investigated in frozen aqueous D2O solutions of 2'-deoxyguanosine (dGuo) at low temperatures at various pHs at which the guanine cation radical, G*+ (pH 3-5), singly deprotonated species, G(-H)* (pH 7-9), and doubly deprotonated species, G(-2H)*- (pH > 11), are found. C-8-deuteration of dGuo to give 8-D-dGuo removes the major proton hyperfine coupling at C-8. This isolates the anisotropic nitrogen couplings for each of the three species and aids our analyses. These anisotropic nitrogen couplings were assigned to specific nitrogen sites by use of 15N-substituted derivatives at N1, N2, and N3 atoms in dGuo. Both ESR and UV-visible spectra are reported for each of the species: G*+, G(-H)*, and G(-2H)*-. The experimental anisotropic ESR hyperfine couplings are compared to those obtained from DFT calculations for the various tautomers of G(-H)*. Using the B3LYP/6-31G(d) method, the geometries and energies of G*+ and its singly deprotonated state in its two tautomeric forms, G(N1-H)* and G(N2-H)*, were investigated. In a nonhydrated state, G(N2-H)* is found to be more stable than G(N1-H)*, but on hydration with seven water molecules G(N1-H)* is found to be more stable than G(N2-H)*. The theoretically calculated hyperfine coupling constants (HFCCs) of G*+, G(N1-H)*, and G(-2H)*- match the experimentally observed HFCCs best on hydration with seven or more waters. For G(-2H)*-, the hyperfine coupling constant (HFCC) at the exocyclic nitrogen atom (N2) is especially sensitive to the number of hydrating water molecules; good agreement with experiment is not obtained until nine or 10 waters of hydration are included.

  1. Hydroxyl Radical (OH•) Reaction with Guanine in an Aqueous Environment: A DFT Study

    Science.gov (United States)

    Kumar, Anil; Pottiboyina, Venkata; Sevilla, Michael D.

    2011-01-01

    The reaction of hydroxyl radical (OH•) with DNA accounts for about half of radiation-induced DNA damage in living systems. Previous literature reports point out that the reaction of OH• with DNA proceeds mainly through the addition of OH• to the C=C bond of the DNA bases. However, recently it has been reported that the principal reaction of OH• with dGuo (deoxyguanosine) is the direct hydrogen atom abstraction from its exocyclic amine group rather than addition of OH• to the C=C bond. In the present work, these two reaction pathways of OH• attack on guanine (G) in the presence of water molecules (aqueous environment) are investigated using the density functional theory (DFT) B3LYP method with 6-31G* and 6-31++G** basis sets. The calculations show that the initial addition of the OH• at C4=C5 double bond of guanine is barrier free and the adduct radical (G-OH•) has only a small activation barrier of ca. 1 – 6 kcal/mol leading to the formation of a metastable ion-pair intermediate (G•+---OH−). The formation of ion-pair is a result of the highly oxidizing nature of the OH• in aqueous media. The resulting ion-pair (G•+---OH−) deprotonates to form H2O and neutral G radicals favoring G(N1-H)• with an activation barrier of ca. 5 kcal/mol. The overall process from the G(C4)-OH• (adduct) to G(N1-H)• and water is found to be exothermic in nature by more than 13 kcal/mol. (G-OH•), (G•+---OH−), and G(N1-H)• were further characterized by the CAM-B3LYP calculations of their UV-visible spectra and good agreement between theory and experiment is achieved. Our calculations for the direct hydrogen abstraction pathway from N1 and N2 sites of guanine by the OH• show that this is also a competitive route to produce G(N2-H)•, G(N1-H)• and H2O. PMID:22050033

  2. Nucleotide sequences encoding a thermostable alkaline protease

    Science.gov (United States)

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  3. The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best.

    Science.gov (United States)

    Zaccaria, F; Paragi, G; Fonseca Guerra, C

    2016-08-21

    The alkali metal ion affinity of guanine quadruplexes has been studied using dispersion-corrected density functional theory (DFT-D). We have done computational investigations in aqueous solution that mimics artificial supramolecular conditions where guanine bases assemble into stacked quartets as well as biological environments in which telomeric quadruplexes are formed. In both cases, an alkali metal cation is needed to assist self-assembly. Our quantum chemical computations on these supramolecular systems are able to reproduce the experimental order of affinity of the guanine quadruplexes for the cations Li(+), Na(+), K(+), Rb(+), and Cs(+). The strongest binding is computed between the potassium cation and the quadruplex as it occurs in nature. The desolvation and the size of alkali metal cations are thought to be responsible for the order of affinity. Until now, the relative importance of these two factors has remained unclear and debated. By assessing the quantum chemical 'size' of the cation, determining the amount of deformation of the quadruplex needed to accommodate the cation and through the energy decomposition analysis (EDA) of the interaction energy between the cation and the guanines, we reveal that the desolvation and size of the alkali metal cation are both almost equally responsible for the order of affinity. PMID:27185388

  4. Thermodynamic potential for the abiotic synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in hydrothermal systems

    OpenAIRE

    Larowe, D. E.; Regnier, P.

    2008-01-01

    The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics of biomolecule synthesis w...

  5. Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

    DEFF Research Database (Denmark)

    Nygaard, Per; Bedsted, Søren; Andersen, Kasper A.K.;

    2000-01-01

    Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in...

  6. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

    Directory of Open Access Journals (Sweden)

    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

  7. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  8. Structural Basis for Bulky-Adduct DNA-Lesion Recognition by the Nucleotide Excision Repair Protein Rad14.

    Science.gov (United States)

    Simon, Nina; Ebert, Charlotte; Schneider, Sabine

    2016-07-25

    Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8-guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion-detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures. PMID:27223336

  9. Mutagenicity associated with O6-methylguanine-DNA damage and mechanism of nucleotide flipping by AGT during repair

    Science.gov (United States)

    Jena, N. R.; Bansal, Manju

    2011-08-01

    Methylated guanine damage at O6 position (i.e. O6MG) is dangerous due to its mutagenic and carcinogenic character that often gives rise to G:C-A:T mutation. However, the reason for this mutagenicity is not known precisely and has been a matter of controversy. Further, although it is known that O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6MG paired with cytosine in DNA, the complete mechanism of target recognition and repair is not known completely. All these aspects of DNA damage and repair have been addressed here by employing high level density functional theory in gas phase and aqueous medium. It is found that the actual cause of O6MG mediated mutation may arise due to the fact that DNA polymerases incorporate thymine opposite to O6MG, misreading the resulting O6MG:T complex as an A:T base pair due to their analogous binding energies and structural alignments. It is further revealed that AGT mediated nucleotide flipping occurs in two successive steps. The intercalation of the finger residue Arg128 into the DNA double helix and its interaction with the O6MG:C base pair followed by rotation of the O6MG nucleotide are found to be crucial for the damage recognition and nucleotide flipping.

  10. Mutagenicity associated with O6-methylguanine-DNA damage and mechanism of nucleotide flipping by AGT during repair

    International Nuclear Information System (INIS)

    Methylated guanine damage at O6 position (i.e. O6MG) is dangerous due to its mutagenic and carcinogenic character that often gives rise to G:C–A:T mutation. However, the reason for this mutagenicity is not known precisely and has been a matter of controversy. Further, although it is known that O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6MG paired with cytosine in DNA, the complete mechanism of target recognition and repair is not known completely. All these aspects of DNA damage and repair have been addressed here by employing high level density functional theory in gas phase and aqueous medium. It is found that the actual cause of O6MG mediated mutation may arise due to the fact that DNA polymerases incorporate thymine opposite to O6MG, misreading the resulting O6MG:T complex as an A:T base pair due to their analogous binding energies and structural alignments. It is further revealed that AGT mediated nucleotide flipping occurs in two successive steps. The intercalation of the finger residue Arg128 into the DNA double helix and its interaction with the O6MG:C base pair followed by rotation of the O6MG nucleotide are found to be crucial for the damage recognition and nucleotide flipping

  11. Sublingual nucleotides and immune response to exercise

    Directory of Open Access Journals (Sweden)

    Ostojic Sergej M

    2012-07-01

    Full Text Available Abstract Evidence exists regarding the potential role of exogenous nucleotides as regulators of the immune function in physically active humans, yet the potential use of nucleotides has been hindered by their low bioavailability after oral administration. We conducted a double-blind, placebo-controlled, randomized trial to assess the effect of sublingual nucleotides (50 mg/day on salivary and serum immunity indicators as compared to placebo, both administered to healthy males aged 20 to 25 years for 14 days. Sublingual administration of nucleotides for 14 days increased serum immunoglobulin A, natural killer cells count and cytotoxic activity, and offset the post-exercise drop of salivary immunoglobulins and lactoferrin (P  0.05. It seems that sublingual administration of nucleotides for two weeks considerably affected immune function in healthy males.

  12. Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads

    Directory of Open Access Journals (Sweden)

    Harikrishnan Jayamohan

    2015-05-01

    Full Text Available In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic beads for capture and polyguanine (polyG oligonucleotide functionalized secondary (polystyrene beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead. While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10\\(^{8}\\ guanine tags per secondary bead (\\(7.5\\times10^{6}\\ biotin-FITC per secondary bead, 20 guanines per oligonucleotide bound to the target (E. coli. A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridineruthenium(II (Ru(bpy\\(_{3}^{2+}\\ as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3

  13. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    Science.gov (United States)

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT.

  14. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.

    Science.gov (United States)

    Yang, C. C.; Oro, J.

    1971-01-01

    Study of the formation of purines, pyrimidines, and other bases from CO, H2, and NH3 under conditions similar to those used in the Fischer-Tropsch process. It is found that industrial nickel/iron alloy catalyzes the synthesis of adenine, guanine, cytosine, and other nitrogenous compounds from mixtures of CO, H2, and NH3 at temperatures of about 600 C. Sufficient sample was accumulated to isolate as solid products adenine, guanine, and cytosine, which were identified by infrared spectrophotometry. In the absence of nickel/iron catalyst, at 650 C, or in the presence of this catalyst, at 450 C, no purines or pyrimidines were synthesized. These results confirm and extend some of the work reported by Kayatsu et al. (1968).

  15. Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks.

    OpenAIRE

    Pereira, T N; Webb, R I; Reilly, P E; Seawright, A. A.; Prakash, A S

    1998-01-01

    Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences. In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as con...

  16. Direct experimental observation of the low ionization potentials of guanine in free oligonucleotides by using photoelectron spectroscopy

    OpenAIRE

    Yang, Xin; Wang, Xue-Bin; Vorpagel, Erich R.; Wang, Lai-Sheng

    2004-01-01

    Photodetachment photoelectron spectroscopy is used to probe the electronic structure of mono-, di-, and trinucleotide anions in the gas phase. A weak and well defined threshold band was observed in the photoelectron spectrum of 2′-deoxyguanosine 5′-monophosphate at a much lower ionization energy than the other three mononucleotides. Density function theory calculations revealed that this unique spectral feature is caused by electron-detachment from a π orbital of the guanine base on 2′-deoxyg...

  17. Role for DNA Polymerase κ in the Processing of N2-N2-Guanine Interstrand Cross-links*S⃞

    OpenAIRE

    Minko, Irina G.; Harbut, Michael B.; Kozekov, Ivan D.; Kozekova, Albena; Jakobs, Petra M.; Olson, Susan B; Moses, Robb E.; Harris, Thomas M.; Rizzo, Carmelo J.; Lloyd, R. Stephen

    2008-01-01

    Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N2-guanines, similar to cross-links formed by mitomycin C and enals. Here, ...

  18. Nucleotide excision repair in the test tube.

    NARCIS (Netherlands)

    N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

    1995-01-01

    textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

  19. Synthesis and Biological Properties of Caffeic Acid-PNA Dimers Containing Guanine

    Directory of Open Access Journals (Sweden)

    Antonio Fiorentino

    2013-07-01

    Full Text Available Caffeic acid (CA; 3,4-dihydroxycinnamic acid is endowed with high antioxidant activity. CA derivatives (such as amides have gained a lot of attention due to their antioxidative, antitumor and antimicrobial properties as well as stable characteristics. Caffeoyl-peptide derivatives showed different antioxidant activity depending on the type and the sequence of amino acid used. For these reasons, we decided to combine CA with Peptide Nucleic Acid (PNA to test whether the new PNA-CA amide derivatives would result in an improvement or gain of CA’s biological (i.e., antioxidant, cytotoxic, cytoprotective properties. We performed the synthesis and characterization of seven dimer conjugates with various combinations of nucleic acid bases and focused NMR studies on the model compound ga-CA dimer. We demonstrate that PNA dimers containing guanine conjugated to CA exhibited different biological activities depending on composition and sequence of the nucleobases. The dimer ag-CA protected HepG2, SK-B-NE(2, and C6 cells from a cytotoxic dose of hydrogen peroxide (H2O2.

  20. Promoter hypermethylation of methyl guanine methyl transferase in lung cancer patients of Kashmir valley

    Directory of Open Access Journals (Sweden)

    Sheikh Mohd Shaffi

    2013-04-01

    Full Text Available Context: MGMT, is a DNA repair protein involved in removing the mutagenic and cytotoxic adducts from O 6 -guanine in DNA, which otherwise can lead to the mutation, primarily due to the tendency of O 6 -methylguanine to pair with thymine during replication, resulting in the conversion of GC to AT pairs, if left unrepaired due to epigenetic silencing of its promoter. Aims: To study the status of MGMT in Kashmiri lung cancer patients Materials and Methods: In this study we analyzed the promoter status of this gene in 50 lung cancer patients using methylation specific-PCR and confirmed by restriction digestion. Results: The promoter was found to be methylated in 52% of the cases, more in male patients (54.2% than in female patients (46.6%. When stratified according to smoking status, current smokers showed a strong association with methylation (OR = 6.0, P = 0.005 than former and never smokers. We also compared the promoter methylation of the MGMT gene with pesticide exposure, and found that patients with pesticide exposure had statistically significant (OR = 7.7 and P = 0.003 association with promoter methylation. Conclusions: Our results indicate that MGMT promoter methylation is associated with smoking exposure and there appears to be an association of MGMT promoter methylation with pesticide exposure in Kashmiri lung cancer patients.

  1. Mutagenic Effects Induced by the Attack of NO2 Radical to the Guanine-Cytosine Base Pair

    Science.gov (United States)

    Cerón-Carrasco, José Pedro; Requena, Alberto; Zúñiga, José; Jacquemin, Denis

    2015-03-01

    We investigate the attack of the nitrogen dioxide radical (NO2) to the guanine-cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC-NO2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis.

  2. Electrodeposition of Prussian Blue Nanoparticles on Electro- chemically Reduced Graphene Oxide and Synergistically Elec- trocatalytic Activity toward Guanine

    Institute of Scientific and Technical Information of China (English)

    杨涛; 关茜; 马苏艳; 李乾和; 焦奎

    2012-01-01

    In this paper, a simple and reliable fabrication method about electrochemically reduced graphene oxide (ERGNO)-prussian blue (PB) nanocomposite was proposed for determination of guanine. Due to its unique struc- tural, physical and chemical properties, ERGNO, which was fabricated on the carbon paste electrode (CPE) before- hand through electrochemical reduction of graphene oxide, was selected as a compatible precursor for next-step PB electrodeposition. Electrochemical behaviors of the resulted PB/ERGNO/CPE were investigated by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The electro- chemical results showed that PB/ERGNO/CPE exhibited good electrochemical performances. The electrocatalytic results of guanine further illustrated that graphene prompted the electrocatalytie ability of PB via the redox shift between PB and prussian yellow (PY) in the potential range from 0.5 to 1.2 V, which has not been widely adopted in the PB based electrochemical sensors. The detection limit of guanine could be calculated to be 1.0 × 10^-8 mol/L. It means this PB/ERGNO/CPE platform is quite sensitive and can be readily applied in biosensor field.

  3. Effects of nucleotides and nucleosides on coagulation

    DEFF Research Database (Denmark)

    Bune, Laurids; Thaning, Pia; Johansson, Pär I;

    2010-01-01

    Nucleotides, including ADP, ATP and uridine triphosphate (UTP), are discharged profusely in the circulation during many pathological conditions including sepsis. Sepsis can cause hypotension and systemic activation of the coagulation and fibrinolytic systems in humans, which may cause disseminated...... intravascular coagulation. We investigated whether nucleotide-induced cardiovascular collapse as provoked by systemic infusion of adenosine, ADP, ATP, UTP and nitric oxide affected the haemostatic system as assessed by whole blood thromboelastography (TEG) analysis. Ten pigs received a randomized infusion...

  4. In vitro incorporation of LNA nucleotides.

    Science.gov (United States)

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product. PMID:18058567

  5. Theoretical Studies on the Interaction between Metal Cations and Cytosine, Guanine

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ya-Ying; ZHOU Li-Xin; WAN Hua-Ping

    2005-01-01

    The interaction of tetra- and hexa-coordinated compounds of cytosine(C) and guanine(G) with metal cations Ca2+, Mg2+, Mn2+, Ni2+, Cu2+, and Zn2+ have been calculated by using the B3LYP/6-31G method at the 6-31G(d, p) basis set, while the remaining coordination bonds are saturated by water molecules ((H2O)4).All geometries were optimized without symmetry restrictions.Comparing the interaction energies we obtained the orders of selectivity of C and G for the above metal ions as follows: aCu2+>aNi2+>aZn2+>aMg2+>bCu2+>aMn2+>bZn2+>bNi2+ and aCu2+> aNi2+>aZn2+>aMg2+>bCu2+>aMn2+>bZn2+>bNi2, respectively (a, b represent tetra- and hexa-coordinated, respectively), which are in good agreement with the experimental facts.Interaction energies of complexes provide a comparatively reliable quantification of the selectivity of dimethyl phosphate anion for the studied metal ions.In addition, the influence of coordination number and coordination structure on the interaction energy and the variation of ionic energy were discussed sufficiently.After analyzing the interaction energies of two kinds of complexes, the "mutual selectivity"as well as the nature of the interaction between metal ions and ligands was revealed.

  6. The use of photosensitizers to selectively generate radiation-induced guanine decomposition products

    Energy Technology Data Exchange (ETDEWEB)

    Buchko, G.W.; Weinfeld, M. [Cross Cancer Inst., Edmonton, Alberta (Canada). Dept. of Radiobiology; Berger, M.; Cadet, J.; Raoul, S. [Centre d`Etudes Nucleaires de Grenoble (France). Lab. des Lesions des Acides Nucleiques

    1995-12-31

    Free radicals generated in vivo through biological processes, including photosensitization, may lead to oxidative damage to cellular DNA. Such photosensitizer-mediated damage to DNA occurs through two principal competitive mechanisms, labeled type 1 and type 2, depending on the process by which an excited photosensitizer decays from its triplet state back to the ground state. Type 1 mechanisms involve initial electron or hydrogen transfer by the excited photosensitizer to, or from, the substrate (DNA) to generate free radicals. Type 2 mechanisms involve the initial generation of singlet oxygen by the excited photosensitizer which subsequently reacts with the substrate to generate unstable peroxidic intermediates. Although in both cases the primary target in DNA is the guanine base, the two mechanisms generate a different set of photoproducts. The lesions 2-amino-5-[(3,5-di-O-acetyl-2-deoxy-{beta}-D-erythro-pentofuran-osyl)-amino]-4H-imidazol-4-one and its major decomposition product, 2,2-diamino-4-[(3,5-di-O-acetyl-2-deoxy-{beta}-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone, have previously been identified and characterized following the exposure of 3{prime},5{prime}-di-O-acetyl-2{prime}-deoxyguanosine to OH radicals in aerated aqueous solution. In this report the authors more fully characterize 2-amino-5-[(2-deoxy-{beta}-D-erythropentofuranosyl)amino]-4H-imidazol-4-one (dIZ) and its major decomposition product, 2,2-diamino-4-[(2-deoxy-{beta}-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (dZ), obtained from the {gamma}-radiolysis of the non-acetyled nucleoside, 2{prime}-deoxyguanosine (dG), in aerated aqueous solution.

  7. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Jett, J. [Lawrence Livermore National Lab., CA (United States)

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  8. Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor.

    Science.gov (United States)

    McDonald, Caleb B; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

    2013-01-01

    Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. PMID:23528987

  9. Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics.

    Science.gov (United States)

    Schaffner-Barbero, Claudia; Gil-Redondo, Rubén; Ruiz-Avila, Laura B; Huecas, Sonia; Läppchen, Tilman; den Blaauwen, Tanneke; Diaz, J Fernando; Morreale, Antonio; Andreu, Jose M

    2010-12-14

    Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

  10. Proofreading of misincorporated nucleotides in DNA transcription.

    Science.gov (United States)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighboring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction. PMID:22643861

  11. Design and synthesis of ATP-based nucleotide analogues and profiling of nucleotide-binding proteins

    NARCIS (Netherlands)

    Wolters, Justina. C.; Roelfes, Johannes; Poolman, Bert

    2011-01-01

    Two nucleotide-based probes were designed and synthesized in order to enrich samples for specific classes of proteins by affinity-based protein profiling. We focused on the profiling of adenine nucleotide-binding proteins. Two properties were considered in the design of the probes: the bait needs to

  12. Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3.

    Science.gov (United States)

    Chandra, Mintu; Datta, Sunando

    2016-08-01

    The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding. PMID:27485554

  13. Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

    Directory of Open Access Journals (Sweden)

    Manjula Pandey

    2014-03-01

    Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

  14. Pan-pathway based interaction profiling of FDA-approved nucleoside and nucleobase analogs with enzymes of the human nucleotide metabolism.

    Directory of Open Access Journals (Sweden)

    Louise Egeblad

    Full Text Available To identify interactions a nucleoside analog library (NAL consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of "off target effects." However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA and uridine phosphorylase 1 (UPP1. An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔT(agg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.

  15. Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release.

    Science.gov (United States)

    Guilfoyle, Amy P; Deshpande, Chandrika N; Schenk, Gerhard; Maher, Megan J; Jormakka, Mika

    2014-01-01

    GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

  16. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    Energy Technology Data Exchange (ETDEWEB)

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  17. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo 184-8588 (Japan); Fujii, Kentaro; Yokoya, Akinari [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki 319-1195 (Japan); Fukuda, Yoshihiro; Saitoh, Yuji [Synchrotron Radiation Research Center, Japan Atomic Energy Agency, Sayo-gun, Hyougo 679-5148 (Japan)

    2014-08-07

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5{sup ′}-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5{sup ′}-monophosphate, and adenosine 5{sup ′}-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  18. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

    Science.gov (United States)

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

    2016-01-22

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

  19. Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

    Science.gov (United States)

    Goupil, Serge; Maréchal, Loïze; El Hajj, Hassan; Tremblay, Marie-Ève; Richard, François J.

    2016-01-01

    In mammals, adenosine 3’, 5’-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE) whose role is to degrade cAMP into 5’-AMP. The PDE superfamily is subdivided into 11 families (PDE1 to 11), which act on either cAMP or cGMP, or on both cAMP and cGMP although with different enzymatic properties. PDE10, which is more effective on cAMP than cGMP, has been known for almost 15 years and is mostly studied in the brain where it is associated with neurological disorders. Although a high level of PDE10A gene expression is observed in the testis, information on the identity of the isoforms or on the cell type that express the PDE10 protein is lacking. The objective of this study was to identify the PDE10A isoforms expressed in the testis and germ cells, and to determine the presence and localization of PDE10A in mature spermatozoa. As a sub-objective, since PDE10A transcript variants were reported strictly through analyses of bovine genomic sequence, we also wanted to determine the nucleotide and amino acid sequences by experimental evidence. Using RT-PCR, 5’- and 3’-RACE approaches we clearly show that PDE10A transcript variants X3 and X5 are expressed in bovine testis as well as in primary spermatocytes and spermatids. We also reveal using a combination of immunological techniques and proteomics analytical tools that the PDE10A isoform X4 is present in the area of the developing acrosome of spermatids and of the acrosome of mature spermatozoa. PMID:27548062

  20. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    Science.gov (United States)

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT. PMID:26968365

  1. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    Science.gov (United States)

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  2. Higher order structural effects stabilizing the reverse watson-crick guanine-cytosine base pair in functional RNAs

    KAUST Repository

    Chawla, Mohit

    2013-10-10

    The G:C reverse Watson-Crick (W:W trans) base pair, also known as Levitt base pair in the context of tRNAs, is a structurally and functionally important base pair that contributes to tertiary interactions joining distant domains in functional RNA molecules and also participates in metabolite binding in riboswitches. We previously indicated that the isolated G:C W:W trans base pair is a rather unstable geometry, and that dicationic metal binding to the Guanine base or posttranscriptional modification of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G:C trans base pairs. Interestingly, 66% of the G:C trans base pairs in the PDB are engaged in additional H-bonding interactions with other bases, the RNA backbone or structured water molecules. High level quantum mechanical calculations on a data set of representative crystal structures were performed to shed light on the structural stability and energetics of the various crystallographic motifs. This analysis was extended to the binding of the preQ1 metabolite to a preQ1-II riboswitch. 2013 The Author(s).

  3. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation

    OpenAIRE

    Yadav, Puja; Owiti, Norah; Kim, Nayun

    2015-01-01

    Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded...

  4. Mitochondrial Adenine Nucleotide Transport and Cardioprotection

    OpenAIRE

    Das, Samarjit; Steenbergen, Charles

    2011-01-01

    Mitochondria are highly metabolically active cell organelles that not only act as the powerhouse of the cell by supplying energy through ATP production, but also play a destructive role by initiating cell death pathways. Growing evidence recognizes that mitochondrial dysfunction is one of the major causes of cardiovascular disease. Under de-energized conditions, slowing of adenine nucleotide transport in and out of the mitochondria significantly attenuates myocardial ischemia-reperfusion inju...

  5. Nucleotide Manipulatives to Illustrate the Central Dogma

    Directory of Open Access Journals (Sweden)

    Sonja B. Yung

    2015-08-01

    Full Text Available The central dogma is a core concept that is critical for introductory biology and microbiology students to master. However, students often struggle to conceptualize the processes involved, and fail to move beyond simply memorizing the basic facts. To encourage critical thinking, we have designed a set of magnetic nucleotide manipulatives that allow students to model DNA structure, along with the processes of replication, transcription, and translation.

  6. Nucleotide Manipulatives to Illustrate the Central Dogma†

    OpenAIRE

    Sonja B. Yung; Todd P. Primm

    2015-01-01

    The central dogma is a core concept that is critical for introductory biology and microbiology students to master. However, students often struggle to conceptualize the processes involved, and fail to move beyond simply memorizing the basic facts. To encourage critical thinking, we have designed a set of magnetic nucleotide manipulatives that allow students to model DNA structure, along with the processes of replication, transcription, and translation.

  7. Visualization of cyclic nucleotide dynamics in neurons

    Directory of Open Access Journals (Sweden)

    Kirill eGorshkov

    2014-12-01

    Full Text Available The second messengers cAMP and cGMP transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks.

  8. Multiphasic interactions between nucleotides and target proteins

    CERN Document Server

    Nissen, Per

    2016-01-01

    The nucleotides guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) bind to target proteins to promote bacterial survival (Corrigan et al. 2016). Thus, the binding of the nucleotides to RsgA, a GTPase, inhibits the hydrolysis of GTP. The dose response, taken to be curvilinear with respect to the logarithm of the inhibitor concentration, is instead much better (P<0.001 when the 6 experiments are combined) represented as multiphasic, with high to exceedingly high absolute r values for the straight lines, and with transitions in the form of non-contiguities (jumps). Profiles for the binding of radiolabeled nucleotides to HprT and Gmk, GTP synthesis enzymes, were, similarly, taken to be curvilinear with respect to the logarithm of the protein concentration. However, the profiles are again much better represented as multiphasic than as curvilinear (the P values range from 0.047 to <0.001 for each of the 8 experiments for binding of ppGpp and pppGpp to HprT). The binding of GTP to HprT and ...

  9. A Sensitive Cyclic Nucleotide Phosphodiesterase Assay for Transient Enzyme Kinetics

    NARCIS (Netherlands)

    Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van

    1983-01-01

    A new assay for cyclic nucleotide phosphodiesterase has been developed by using reverse-phase column chromatography for the separation of product and substrate of the enzymatic reaction. The polar 5'-nucleotides are not retarded by the column, while the more lipophilic cyclic nucleotides bind to the

  10. Highly sensitive sensor for picomolar detection of insulin at physiological pH, using GC electrode modified with guanine and electrodeposited nickel oxide nanoparticles.

    Science.gov (United States)

    Salimi, Abdollah; Noorbakhash, Abdollah; Sharifi, Ensieh; Semnani, Abolfazl

    2008-12-01

    The electrochemical behavior of insulin at glassy carbon (GC) electrode modified with nickel oxide nanoparticles and guanine was investigated. Cyclic voltammetry technique has been used for electrodeposition of nickel oxide nanoparticles (NiOx) and immobilization of guanine on the surface GC electrode. In comparison to glassy carbon electrode modified with nickel oxide nanoparticles and bare GC electrode modified with adsorbed guanine, the guanine/nickel oxide nanoparticles/modified GC electrode exhibited excellent catalytic activity for the oxidation of insulin in physiological pH solutions at reduced overpotential. The modified electrode was applied for insulin detection using cyclic voltammetry or hydrodynamic amperometry techniques. It was found that the calibration curve was linear up to 4muM with a detection limit of 22pM and sensitivity of 100.9pA/pM under the optimized condition for hydrodynamic amperometry using a rotating disk modified electrode. In comparison to other electrochemical insulin sensors, this sensor shows many advantages such as simple preparation method without using any special electron transfer mediator or specific reagent, high sensitivity, excellent catalytic activity at physiological pH values, short response time, long-term stability and remarkable antifouling property toward insulin and its oxidation product. Additionally, it is promising for the monitoring of insulin in chromatographic effluents.

  11. Quantum processes in 8-Oxo-Guanine-Ru(bipyridine)32+ photosynthetic systems of artificial minimal cells

    Science.gov (United States)

    Tamulis, Arvydas; Grigalavicius, Mantas; Krisciukaitis, Sarunas; Medzevicius, Giedrius

    2011-06-01

    Density functional theory methods were used to investigate various self-assembled photoactive bioorganic systems of interest for artificial minimal cells. The cell systems studied are based on nucleotides or their compounds and consisted of up to 123 atoms (not including the associated water or methanol solvent shells) and are up to 2.5 nm in diameter. The electron correlation interactions responsible for the weak hydrogen and Van derWaals chemical bonds increase due to the addition of a polar solvent (water or methanol). The precursor fatty acid molecules of the system also play a critical role in the quantum mechanical interaction based self-assembly of the photosynthetic center and the functioning of the photosynthetic processes of the artificial minimal cells. The distances between the separated sensitizer, fatty acid precursor, and methanol molecules are comparable to Van derWaals and hydrogen bonding radii. As a result the associated electron correlation interactions compress the overall system, resulting in an even smaller gap between the highest occupied molecular orbital (HOMO), and lowest unoccupied molecular orbital (LUMO) electron energy levels and photoexcited electron tunnelling occurs from the sensitizer (either Ru(bpy)32+ or [Ru(bpy)2(4-Bu-4'-Me-2,2'-bpy)]2++ derivatives) to the precursor fatty acid molecules (notation used: Me = methyl; Bu = butyl; bpy = bipyridine). The shift of the absorption spectrum to the red for the artificial protocell photosynthetic centers might be considered as the measure of the complexity of these systems.

  12. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Science.gov (United States)

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  13. A colorimetric method for the determination of lead(II) ions using gold nanoparticles and a guanine-rich oligonucleotide

    International Nuclear Information System (INIS)

    We have developed a colorimetric method for the determination of Pb(II) ions. It is based on the use of gold nanoparticles and a guanine-rich synthetic oligonucleotide. On addition of Pb(II), the color of the solution turns from red to blue. The ratio of the UV-vis absorption at 630 nm and 525 nm is proportional to the concentration of Pb(II) ions in the range from 10 to 100 nM, and the detection limit is 20 nM. Other metal ions do not interfere if present in up to a 10-fold molar excess. The method was successfully applied to the detection of Pb(II) in lake water and urine. The recovery in case of spiked samples is 92%. The results show that this method is sensitive, simple and fast. (author)

  14. Atomic-Scale Insight into Tautomeric Recognition, Separation, and Interconversion of Guanine Molecular Networks on Au(111).

    Science.gov (United States)

    Zhang, Chi; Xie, Lei; Wang, Likun; Kong, Huihui; Tan, Qinggang; Xu, Wei

    2015-09-16

    Although tautomerization may directly affect the chemical or biological properties of molecules, real-space investigation on the tautomeric behaviors of organic molecules in a larger area of molecular networks has been scarcely reported. In this paper, we choose guanine (G) molecule as a model system. From the interplay of high-resolution scanning tunneling microscopy (STM) imaging and density functional theory (DFT) calculations, we have successfully achieved the tautomeric recognition, separation, and interconversion of G molecular networks (formed by two tautomeric forms G/9H and G/7H) with the aid of NaCl on the Au(111) surface in ultrahigh vacuum (UHV) conditions. Our results may serve as a prototypical system to provide important insights into tautomerization related issues, which should be intriguing to biochemistry, pharmaceutics, and other related fields. PMID:26322860

  15. Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk.

    Science.gov (United States)

    Lewin, Michelle H; Bailey, Nina; Bandaletova, Tanya; Bowman, Richard; Cross, Amanda J; Pollock, Jim; Shuker, David E G; Bingham, Sheila A

    2006-02-01

    Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N-nitrosocompounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, high-fiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O(6)-carboxymethyl guanine (O(6)CMG) was significantly (P colorectal cancer. PMID:16452248

  16. A single nucleotide polymorphism in NEUROD1 is associated with production traits in Nelore beef cattle.

    Science.gov (United States)

    de Oliveira, P S N; Tizioto, P C; Malago, W; do Nascimento, M L; Cesar, A S M; Diniz, W J S; de Souza, M M; Lanna, D P D; Tullio, R R; Mourão, G B; de A Mudadu, M; Coutinho, L L; de A Regitano, L C

    2016-01-01

    Feed efficiency and carcass characteristics are late-measured traits. The detection of molecular markers associated with them can help breeding programs to select animals early in life, and to predict breeding values with high accuracy. The objective of this study was to identify polymorphisms in the functional and positional candidate gene NEUROD1 (neurogenic differentiation 1), and investigate their associations with production traits in reference families of Nelore cattle. A total of 585 steers were used, from 34 sires chosen to represent the variability of this breed. By sequencing 14 animals with extreme residual feed intake (RFI) values, seven single nucleotide polymorphisms (SNPs) in NEUROD1 were identified. The investigation of marker effects on the target traits RFI, backfat thickness (BFT), ribeye area (REA), average body weight (ABW), and metabolic body weight (MBW) was performed with a mixed model using the restricted maximum likelihood method. SNP1062, which changes cytosine for guanine, had no significant association with RFI or REA. However, we found an additive effect on ABW (P ≤ 0.05) and MBW (P ≤ 0.05), with an estimated allele substitution effect of -1.59 and -0.93 kg0.75, respectively. A dominant effect of this SNP for BFT was also found (P ≤ 0.010). Our results are the first that identify NEUROD1 as a candidate that affects BFT, ABW, and MBW. Once confirmed, the inclusion of this SNP in dense panels may improve the accuracy of genomic selection for these traits in Nelore beef cattle as this SNP is not currently represented on SNP chips. PMID:27420997

  17. Echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTH1

    Directory of Open Access Journals (Sweden)

    Dong L

    2015-12-01

    Full Text Available Liwei Dong,1 Hongge Wang,1 Jiajing Niu,1 Mingwei Zou,2 Nuoting Wu,1 Debin Yu,1 Ye Wang,1 Zhihua Zou11Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China; 2Department of Psychology, College of Liberal Arts and Social Sciences, University of Houston, Houston, TX, USA Abstract: Inhibition of the nucleotide pool sanitizing enzyme MTH1 causes extensive oxidative DNA damages and apoptosis in cancer cells and hence may be used as an anticancer strategy. As natural products have been a rich source of medicinal chemicals, in the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in vitro screening assay to search for natural compounds capable of inhibiting MTH1. Echinacoside, a compound derived from the medicinal plants Cistanche and Echinacea, effectively inhibited the catalytic activity of MTH1 in an in vitro assay. Treatment of various human cancer cell lines with Echinacoside resulted in a significant increase in the cellular level of oxidized guanine (8-oxoguanine, while cellular reactive oxygen species level remained unchanged, indicating that Echinacoside also inhibited the activity of cellular MTH1. Consequently, Echinacoside treatment induced an immediate and dramatic increase in DNA damage markers and upregulation of the G1/S-CDK inhibitor p21, which were followed by marked apoptotic cell death and cell cycle arrest in cancer but not in noncancer cells. Taken together, these studies identified a natural compound as an MTH1 inhibitor and suggest that natural products can be an important source of anticancer agents. Keywords: Echinacoside, MTH1, 8-oxoG, DNA damage, apoptosis, cell cycle arrest

  18. Nucleotide Excision Repair in Caenorhabditis elegans

    OpenAIRE

    Hannes Lans; Wim Vermeulen

    2011-01-01

    Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vi...

  19. Performance characteristics of guanine incorporated PVDF-HFP/PEO polymer blend electrolytes with binary iodide salts for dye-sensitized solar cells

    Science.gov (United States)

    Senthil, R. A.; Theerthagiri, J.; Madhavan, J.; Arof, A. K.

    2016-08-01

    In this work, we have investigated the influence of guanine as an organic dopant in dye-sensitized solar cell (DSSC) based on poly(vinylidinefluoride-co-hexafluoropropylene) (PVDF-HFP)/polyethylene oxide (PEO) polymer blend electrolyte along with binary iodide salts (potassium iodide (KI) and tetrabutylammonium iodide (TBAI)) and iodine (I2). The PVDF-HFP/KI + TBAI/I2, PVDF-HFP/PEO/KI + TBAI/I2 and guanine incorporated PVDF-HFP/PEO/KI + TBAI/I2 electrolytes were prepared by solution casting technique using DMF as solvent. The PVDF-HFP/KI + TBAI/I2 electrolyte showed an ionic conductivity value of 9.99 × 10-5 Scm-1, whereas, it was found to be increased to 4.53 × 10-5 Scm-1 when PEO was blended with PVDF-HFP/KI + TBAI/I2 electrolyte. However, a maximum ionic conductivity value of 3.67 × 10-4 Scm-1 was obtained for guanine incorporated PVDF-HFP/PEO/KI + TBAI/I2 blend electrolyte. The photovoltaic properties of all these polymer electrolytes in DSSCs were characterized. As a consequence, the power conversion efficiency of the guanine incorporated PVDF-HFP/PEO/KI + TBAI/I2 electrolyte based DSSC was significantly improved to 4.98% compared with PVDF-HFP/PEO/KI + TBAI/I2 electrolyte based DSSC (2.46%). These results revealed that the guanine can be an effective organic dopant to enhance the performance of DSSCs.

  20. A novel nuclear role for the Vav3 nucleotide exchange factor in androgen receptor coactivation in prostate cancer.

    Science.gov (United States)

    Rao, S; Lyons, L S; Fahrenholtz, C D; Wu, F; Farooq, A; Balkan, W; Burnstein, K L

    2012-02-01

    Increased androgen receptor (AR) transcriptional activity mediated by coactivator proteins may drive castration-resistant prostate cancer (CRPC) growth. Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is overexpressed in human prostate cancers, particularly in models of CRPC progression. Vav3 coactivates AR in a Vav3 pleckstrin homology (PH) domain-dependent but GEF-independent manner. Ectopic expression of Vav3 in androgen-dependent human prostate cancer cells conferred robust castration-resistant xenograft tumor growth. Vav3 but not a Vav3 PH mutant greatly stimulated interaction between the AR amino and carboxyl termini (N-C interaction), which is required for maximal receptor transcriptional activity. Vav3 was distributed between the cytoplasm and nucleus with nuclear localization-dependent on the Vav3 PH domain. Membrane targeting of Vav3 abolished Vav3 potentiation of AR activity, whereas nuclear targeting of a Vav3 PH mutant rescued AR coactivation, suggesting that nuclear localization is an important function of the Vav3 PH domain. A nuclear role for Vav3 was further demonstrated by sequential chromatin immunoprecipitation assays, which revealed that Vav3 and AR were recruited to the same transcriptional complexes of an AR target gene enhancer. These data demonstrate the importance of Vav3 in CRPC and define a novel nuclear function of Vav3 in regulating AR activity.

  1. Single nucleotide polymorphism of prolactin gene exon two in ducks of Pekin, Mojosari and Pekin Mojosari crossbred

    Directory of Open Access Journals (Sweden)

    Irma

    2014-05-01

    Full Text Available Prolactin gene plays crucial role in the reproduction and egg production of birds. The objectives of this study were to characterize single nucleotide polymorphism in partial intron and coding region of duck prolactin gene. Blood samples were collected from 168 ducks consisted of 19 Pekin, 36 Mojosari, and 113 of their crossbreds collected from Indonesian Research Institute for Animal Production (IRIAP. Primer pairs for the coding regions in prolactin gene were self designed based on the duck genomic sequence database (GeneBank: AB158611.1. PCR products based on DNA of prolactin gene exon two was amplified approximately 400 bp. There is one base insertion of Adenin at the position of 2001 bp intron two region of duck prolactin. Homology test based on BLAST method indicated 99% identity with duck refference (Code Access GeneBank: AB158611.1. Adenin composition in all of duck samples was higher than refference. Triplet hydrogen bonds between Guanine and Cytosin pairs was higher than those at duplet hydrogen bonds between Adenine and Thymine. All duck samples were homozigous and monomorphyc.

  2. Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

    OpenAIRE

    Wright, G E; Baril, E F; Brown, V M; Brown, N C

    1982-01-01

    The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, w...

  3. A computational study of a recreated G protein-GEF reaction intermediate competent for nucleotide exchange: fate of the Mg ion.

    Directory of Open Access Journals (Sweden)

    Mériam Ben Hamida-Rebaï

    Full Text Available Small G-proteins of the superfamily Ras function as molecular switches, interacting with different cellular partners according to their activation state. G-protein activation involves the dissociation of bound GDP and its replacement by GTP, in an exchange reaction that is accelerated and regulated in the cell by guanine-nucleotide exchange factors (GEFs. Large conformational changes accompany the exchange reaction, and our understanding of the mechanism is correspondingly incomplete. However, much knowledge has been derived from structural studies of blocked or inactive mutant GEFs, which presumably closely represent intermediates in the exchange reaction and yet which are by design incompetent for carrying out the nucleotide exchange reaction. In this study we have used comparative modelling to recreate an exchange-competent form of a late, pre-GDP-ejection intermediate species in Arf1, a well-characterized small G-protein. We extensively characterized three distinct models of this intermediate using molecular dynamics simulations, allowing us to address ambiguities related to the mutant structural studies. We observed in particular the unfavorable nature of Mg2+ associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence. The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction. The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.

  4. Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

    Science.gov (United States)

    Endrizzi, James A; Kim, Hanseong; Anderson, Paul M; Baldwin, Enoch P

    2004-06-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

  5. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao

    2011-01-01

    Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms.While bacteria require only three proteins to complete the incision step of NER,eukaryotes employ about 30 proteins to complete the same step.Here we summarize recent studies demonstrating that ubiquitination,a post-translational modification,plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis.Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process.We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

  6. Structure of the Hsp110:Hsc70 Nucleotide Exchange Machine

    OpenAIRE

    Schuermann, Jonathan P.; Jiang, Jianwen; Cuellar, Jorge; Llorca, Oscar; Wang, Liping; Gimenez, Luis E.; Jin, Suping; Taylor, Alexander B.; Demeler, Borries; Morano, Kevin A.; Hart, P. John; Valpuesta, Jose M.; Lafer, Eileen M.; Sousa, Rui

    2008-01-01

    Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging...

  7. Frequency and Correlation of Nearest Neighboring Nucleotides in Human Genome

    Science.gov (United States)

    Jin, Neng-zhi; Liu, Zi-xian; Qiu, Wen-yuan

    2009-02-01

    Zipf's approach in linguistics is utilized to analyze the statistical features of frequency and correlation of 16 nearest neighboring nucleotides (AA, AC, AG, ..., TT) in 12 human chromosomes (Y, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, and 12). It is found that these statistical features of nearest neighboring nucleotides in human genome: (i) the frequency distribution is a linear function, and (ii) the correlation distribution is an inverse function. The coefficients of the linear function and inverse function depend on the GC content. It proposes the correlation distribution of nearest neighboring nucleotides for the first time and extends the descriptor about nearest neighboring nucleotides.

  8. Multiscale QM/MM Molecular Dynamics Study on the First Steps of Guanine-Damage by Free Hydroxyl Radicals in Solution

    CERN Document Server

    Abolfath, Ramin M; Rajnarayanam, R; Brabec, Thomas; Kodym, Reinhard; Papiez, Lech

    2012-01-01

    Understanding the damage of DNA bases from hydrogen abstraction by free OH radicals is of particular importance to reveal the effect of hydroxyl radicals produced by the secondary effect of radiation. Previous studies address the problem with truncated DNA bases as ab-initio quantum simulation required to study such electronic spin dependent processes are computationally expensive. Here, for the first time, we employ a multiscale and hybrid Quantum-Mechanical-Molecular-Mechanical simulation to study the interaction of OH radicals with guanine-deoxyribose-phosphate DNA molecular unit in the presence of water where all the water molecules and the deoxyribose-phosphate fragment are treated with the simplistic classical Molecular-Mechanical scheme. Our result illustrates that the presence of water strongly alters the hydrogen-abstraction reaction as the hydrogen bonding of OH radicals with water restricts the relative orientation of the OH-radicals with respective to the the DNA base (here guanine). This results ...

  9. mRNA:guanine-N7 cap methyltransferases: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships

    OpenAIRE

    Radlinska Monika; Feder Marcin; Bujnicki Janusz M; Rychlewski Leszek

    2001-01-01

    Abstract Background The 5'-terminal cap structure plays an important role in many aspects of mRNA metabolism. Capping enzymes encoded by viruses and pathogenic fungi are attractive targets for specific inhibitors. There is a large body of experimental data on viral and cellular methyltransferases (MTases) that carry out guanine-N7 (cap 0) methylation, including results of extensive mutagenesis. However, a crystal structure is not available and cap 0 MTases are too diverged from other MTases o...

  10. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    Directory of Open Access Journals (Sweden)

    Linder Markus

    2006-03-01

    Full Text Available Abstract Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range

  11. Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types.

    Science.gov (United States)

    Momose, Fumiyasu; Seo, Naohiro; Akahori, Yasushi; Sawada, Shin-Ichi; Harada, Naozumi; Ogura, Toru; Akiyoshi, Kazunari; Shiku, Hiroshi

    2016-01-01

    Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases. PMID:27101102

  12. Evidence that Natural Selection is the Primary Cause of the Guanine-cytosine Content Variation in Rice Genes

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Shi; Xiyin Wang; Zhe Li; Qihui Zhu; Ji Yang; Song Ge; Jingchu Luo

    2007-01-01

    Cereal genes are classified into two distinct classes according to the guanine-cytosine (GC) content at the third codon sites (GC3). Natural selection and mutation bias have been proposed to affect the GC content. However, there has been controversy about the cause of GC variation. Here, we characterized the GC content of 1 092 paralogs and other single-copy genes in the duplicated chromosomal regions of the rice genome (ssp. indica) and classified the paralogs into GC3-rich and GC3-poor groups. By referring to out-group sequences from Arabidopsis and maize, we confirmed that the average synonymous substitution rate of the GC3-rich genes is significantly lower than that of the GC3-poor genes. Furthermore,we explored the other possible factors corresponding to the GC variation including the length of coding sequences, the number of exons in each gene, the number of genes in each family, the location of genes on chromosomes and the protein functions. Consequently, we propose that natural selection rather than mutation bias was the primary cause of the GC variation.

  13. Crystalline guanine adducts of natural and synthetic trioxacarcins suggest a common biological mechanism and reveal a basis for the instability of trioxacarcin A.

    Science.gov (United States)

    Pröpper, Kevin; Dittrich, Birger; Smaltz, Daniel J; Magauer, Thomas; Myers, Andrew G

    2014-09-15

    X-ray crystallographic characterization of products derived from natural and fully synthetic trioxacarcins, molecules with potent antiproliferative effects, illuminates aspects of their reactivity and mechanism of action. Incubation of the fully synthetic trioxacarcin analog 3, which lacks one of the carbohydrate residues present in the natural product trioxacarcin A (1) as well as oxygenation at C2 and C4 yet retains potent antiproliferative effects, with the self-complimentary duplex oligonucleotide d(AACCGGTT) led to production of a crystalline covalent guanine adduct (6). Adduct 6 is closely analogous to gutingimycin (2), the previously reported guanine adduct derived from incubation of natural trioxacarcin A (1) with duplex DNA, suggesting that 3 and 1 likely share a common basis of cytotoxicity. In addition, we isolated a novel, dark-red crystalline guanine adduct (7) from incubation of trioxacarcin A itself with the self-complimentary duplex oligonucleotide d(CGTATACG). Crystallographic analysis suggests that 7 is an anthraquinone derivative, which we propose arises by a sequence of guanosine alkylation within duplex DNA, depurination, base-catalyzed elimination of the trioxacarcinose A carbohydrate residue, and oxidative rearrangement to form an anthraquinone. We believe that this heretofore unrecognized chemical instability of natural trioxacarcins may explain why trioxacarcin analogs lacking C4 oxygenation exhibit superior chemical stabilities yet, as evidenced by structure 3, retain a capacity to form lesions with duplex DNA. PMID:25176186

  14. Nucleotide sequence of cloned rat serum albumin messenger RNA.

    OpenAIRE

    Sargent, T D; Yang, M; Bonner, J.

    1981-01-01

    The nucleotide sequences of the recombinant DNA inserts of three bacterial plasmid clones containing nearly all of the rat serum albumin mRNA have been determined. A statistical analysis of the nucleotide sequence reveals a pattern of repeated internal homology that confirms the "intragenic triplication" model of albumin evolution.

  15. Prebiotic nucleotide synthesis demonstration of a geologically plausible pathway

    NARCIS (Netherlands)

    Schwartz, A.W.; Veen, van der M.; Bisseling, T.; Chittenden, G.J.

    1975-01-01

    Mineral phosphate (apatite) is activated for the synthesis of nucleotides when dilute solutions containing nucleoside and ammonium oxalate are evaporated in its presence. A natural, igneous fluorapatite was found to be even more effective in nucleotide synthesis than the more soluble hydroxylapatite

  16. Association study of nonsynonymous single nucleotide polymorphisms in schizophrenia

    DEFF Research Database (Denmark)

    Carrera, Noa; Arrojo, Manuel; Sanjuán, Julio;

    2012-01-01

    Genome-wide association studies using several hundred thousand anonymous markers present limited statistical power. Alternatively, association studies restricted to common nonsynonymous single nucleotide polymorphisms (nsSNPs) have the advantage of strongly reducing the multiple testing problem......, while increasing the probability of testing functional single nucleotide polymorphisms (SNPs)....

  17. Nucleotide Excision Repair in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Hannes Lans

    2011-01-01

    Full Text Available Nucleotide excision repair (NER plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage.

  18. Software for tag single nucleotide polymorphism selection

    Directory of Open Access Journals (Sweden)

    Stram Daniel O

    2005-06-01

    Full Text Available Abstract This paper reviews the theoretical basis for single nucleotide polymorphism (SNP tagging and considers the use of current software made freely available for this task. A distinction between haplotype block-based and non-block-based approaches yields two classes of procedures. Analysis of two different sets of SNP genotype data from the HapMap is used to judge the practical aspects of using each of the programs considered, as well as to make some general observations about the performance of the programs in finding optimal sets of tagging SNPs. Pairwise R2 methods, while the simplest of those considered, do tend to pick more tagging SNPs than are strictly needed to predict unmeasured (non-tagging SNPs, since a combination of two or more tagging SNPs can form a prediction of SNPs that have no direct (pairwise surrogate. Block-based methods that exploit the linkage disequilibrium structure within haplotype blocks exploit this sort of redundancy, but run a risk of over-fitting if used without some care. A compromise approach which eliminates the need first to analyse block structure, but which still exploits simple relationships between SNPs, appears promising.

  19. Empirical Bayes analysis of single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Ickstadt Katja

    2008-03-01

    Full Text Available Abstract Background An important goal of whole-genome studies concerned with single nucleotide polymorphisms (SNPs is the identification of SNPs associated with a covariate of interest such as the case-control status or the type of cancer. Since these studies often comprise the genotypes of hundreds of thousands of SNPs, methods are required that can cope with the corresponding multiple testing problem. For the analysis of gene expression data, approaches such as the empirical Bayes analysis of microarrays have been developed particularly for the detection of genes associated with the response. However, the empirical Bayes analysis of microarrays has only been suggested for binary responses when considering expression values, i.e. continuous predictors. Results In this paper, we propose a modification of this empirical Bayes analysis that can be used to analyze high-dimensional categorical SNP data. This approach along with a generalized version of the original empirical Bayes method are available in the R package siggenes version 1.10.0 and later that can be downloaded from http://www.bioconductor.org. Conclusion As applications to two subsets of the HapMap data show, the empirical Bayes analysis of microarrays cannot only be used to analyze continuous gene expression data, but also be applied to categorical SNP data, where the response is not restricted to be binary. In association studies in which typically several ten to a few hundred SNPs are considered, our approach can furthermore be employed to test interactions of SNPs. Moreover, the posterior probabilities resulting from the empirical Bayes analysis of (prespecified interactions/genotypes can also be used to quantify the importance of these interactions.

  20. The housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT regulates multiple developmental and metabolic pathways of murine embryonic stem cell neuronal differentiation.

    Directory of Open Access Journals (Sweden)

    Tae Hyuk Kang

    Full Text Available The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT cause the severe neurodevelopmental Lesch Nyhan Disease (LND are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease.

  1. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    Institute of Scientific and Technical Information of China (English)

    Wei Liao; Sanjai Sharma

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells.

  2. (/sup 3/H)dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states

    Energy Technology Data Exchange (ETDEWEB)

    Hamblin, M.W.; Ariani, K.; Adriaenssens, P.I.; Ciaranello, R.D.

    1987-12-01

    (/sup 3/H)Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation (Bmax) = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of (/sup 3/H)DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, (/sup 3/H)DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. (/sup 3/H)DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of (/sup 3/H)DE.

  3. Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

    Directory of Open Access Journals (Sweden)

    Simon Reed

    2012-09-01

    Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

  4. Frequency and Correlation of Nearest Neighboring Nucleotides in Human Genome

    Institute of Scientific and Technical Information of China (English)

    Neng-zhi Jin; Zi-xian Liu; Wen-yuan Qiu

    2009-01-01

    Zipf's approach in linguistics is utilized to analyze the statistical features of frequency and mosomes (Y, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, and 12). It is found that these statistical features of nearest neighboring nucleotides in human genome: (ⅰ) the frequency distribution is a linear function, and (ⅱ) the correlation distribution is an inverse function. The coeffi-cients of the linear function and inverse function depend on the GC content. It proposes the correlation distribution of nearest neighboring nucleotides for the first time and extends the descriptor about nearest neighboring nucleotides.

  5. Overview of the application of nucleotide in aquaculture

    Directory of Open Access Journals (Sweden)

    Hoang Do Huu

    2016-10-01

    Full Text Available Although long history application in infant formula, dietary nucleotide supplementation has been used only recently in the evaluation of growth performance, stress and pathogen resistance in aquaculture species. This paper addresses the present knowledge of the use of nucleotide supplemented in the diet for culture species. Research reveals that dietary nucleotide may have significant impact and is recommended to add to the feed of aquatic species to get better performance. However, more studies should also be conducted to have better understandings on dose requirement, duration of application, impact on different life stage and under different environmental stress and pathogens. Further study should also examine the effects of dietary nucleotide supplementation of intestinal microbiota and gut morphology, and immune response of aquaculture species.

  6. Nucleotide Metabolism and its Control in Lactic Acid Bacteria

    DEFF Research Database (Denmark)

    Kilstrup, Mogens; Hammer, Karin; Jensen, Peter Ruhdal;

    2005-01-01

    the interconversion pathways, the formation of deoxyribonucleotides and the salvage pathways for use of exogenous precursors. The data for the enzymatic and the genetic regulation of these pathways are reviewed, as well as the gene organizations in different lactic acid bacteria. Mutant phenotypes and methods...... for manipulation of nucleotide pools are also discussed. Our aim is to provide an overview of the physiology and genetics of nucleotide metabolism and its regulation that will facilitate the interpretation of data arising from genetics, metabolomics, proteomics, and transcriptomics in lactic acid bacteria.......Most metabolic reactions are connected through either their utilization of nucleotides or their utilization of nucleotides or their regulation by these metabolites. In this review the biosynthetic pathways for pyrimidine and purine metabolism in lactic acid bacteria are described including...

  7. Single nucleotide polymorphism (SNP) detection on a magnetoresistive sensor

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Østerberg, Frederik Westergaard; Dufva, Martin;

    2013-01-01

    We present a magnetoresistive sensor platform for hybridization assays and demonstrate its applicability on single nucleotide polymorphism (SNP) genotyping. The sensor relies on anisotropic magnetoresistance in a new geometry with a local negative reference and uses the magnetic field from...

  8. Tissue-specific accelerated aging in nucleotide excision repair deficiency

    OpenAIRE

    Laura J. Niedernhofer

    2008-01-01

    Nucleotide excision repair (NER) is a multi-step DNA repair mechanism that removes helix-distorting modified nucleotides from the genome. NER is divided into two subpathways depending on the location of DNA damage in the genome and how it is first detected. Global genome NER identifies and repairs DNA lesions throughout the genome. This subpathway of NER primarily protects against the accumulation of mutations in the genome. Transcription-coupled (TC) NER rapidly repairs lesions in the transc...

  9. Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides

    OpenAIRE

    Pasternak, Anna; Wengel, Jesper

    2010-01-01

    Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4...

  10. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Directory of Open Access Journals (Sweden)

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  11. Nucleotide-sugar transporters: structure, function and roles in vivo

    Directory of Open Access Journals (Sweden)

    Handford M.

    2006-01-01

    Full Text Available The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs. These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

  12. Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

    OpenAIRE

    Buck, Janina; Noeske, Jonas; Wöhnert, Jens; Schwalbe, Harald

    2010-01-01

    Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) result...

  13. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    Science.gov (United States)

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

  14. The tail domain of myosin M catalyses nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions.

    Science.gov (United States)

    Geissler, H; Ullmann, R; Soldati, T

    2000-05-01

    Members of the myosin superfamily play crucial roles in cellular processes including management of the cortical cytoskeleton, organelle transport and signal transduction. GTPases of the Rho family act as key control elements in the reorganization of the actin cytoskeleton in response to growth factors, and other functions such as membrane trafficking, transcriptional regulation, growth control and development. Here, we describe a novel unconventional myosin from Dictyostelium discoideum, MyoM. Primary sequence analysis revealed that it has the appearance of a natural chimera between a myosin motor domain and a guanine nucleotide exchange factor (GEF) domain for Rho GTPases. The functionality of both domains was established. Binding of the motor domain to F-actin was ATP-dependent and potentially regulated by phosphorylation. The GEF domain displayed selective activity on Rac1-related GTPases. Overexpression, rather than absence of MyoM, affected the cell morphology and viability. Particularly in response to hypo-osmotic stress, cells overexpressing the MyoM tail domain extended massive actin-driven protrusions. The GEF was enriched at the tip of growing protuberances, probably through its pleckstrin homology domain. MyoM is the first unconventional myosin containing an active Rac-GEF domain, suggesting a role at the interface of Rac-mediated signal transduction and remodeling of the actin cytoskeleton. PMID:11208126

  15. Causes and consequences of plant radio-resistance. Formation of DNA basis lesions and self-repairing activity of one of them, the 8-oxo-7,8-dihydro-guanine in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    In this research thesis, the author first explains how and why DNA is injured when it is submitted to an oxidizing stress, and describes precisely the formation and the biological consequences of lesions of DNA bases, the 8-oxo-7,8-dihydro-guanine (8-oxoGua). She describes the repairing activities of the oxidized DNA, and more particularly the repairing of 8-oxoGua, in prokaryotes as well as in yeast, mammals and plants. Methodologies used are described, together with the repair activities of the 8-oxo-7,8-dihydro-guanine following a biochemical type approach and a molecular biology approach

  16. Palladium-Catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Kevin H. Shaughnessy

    2015-05-01

    Full Text Available Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

  17. Dynamics of Charge Transfer in Ordered and Chaotic Nucleotide Sequences

    CERN Document Server

    Fialko, N S

    2013-01-01

    Charge transfer is considered in systems composed of a donor, an acceptor and bridge sites of (AT) nucleotide pairs. For a bridge consisting of 180 (AT) pairs, three cases are dealt with: a uniform case, when all the nucleotides in each strand are identical; an ordered case, when nucleotides in each DNA strand are arranged in an orderly fashion; a chaotic case, when (AT) and (TA) pairs are arranged randomly. It is shown that in all the cases a charge transfer from a donor to an acceptor can take place. All other factors being equal, the transfer is the most efficient in the uniform case, the ordered and chaotic cases are less and the least efficient, accordingly. The results obtained are in agreement with experimental data on long-range charge transfer in DNA.

  18. Fluorescence chemosensors with pyrene and their interaction with nucleotide phosphate

    Institute of Scientific and Technical Information of China (English)

    李华平; 汪鹏飞; 吴世康

    1999-01-01

    A group of fluorescence chemosensor with pyrene, compounds (Ⅰ), (Ⅱ) and (Ⅲ), were synthesized The fluorescence spectra and the lifetime of these compounds were carefully measured. The fluorescence quenching spec tra of pyrenyl butyric acid, compounds (Ⅰ), (Ⅱ) and (Ⅲ) by different nucleotide phosphates, AMP ADP, ATP dTTP, were also recorded and studied. The quenching and the stability constants were calculated by Stern-Volmer equa tion and eq. (2), respectively. The mechanism of interaction between fluorescence chemosensor and nucleotide phos phate was didscussed based on the comparison of the results obtained with the CPK model of free molecules of these com pounds in the ground state.

  19. Biocuration of functional annotation at the European nucleotide archive.

    Science.gov (United States)

    Gibson, Richard; Alako, Blaise; Amid, Clara; Cerdeño-Tárraga, Ana; Cleland, Iain; Goodgame, Neil; Ten Hoopen, Petra; Jayathilaka, Suran; Kay, Simon; Leinonen, Rasko; Liu, Xin; Pallreddy, Swapna; Pakseresht, Nima; Rajan, Jeena; Rosselló, Marc; Silvester, Nicole; Smirnov, Dmitriy; Toribio, Ana Luisa; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy

    2016-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is a repository for the submission, maintenance and presentation of nucleotide sequence data and related sample and experimental information. In this article we report on ENA in 2015 regarding general activity, notable published data sets and major achievements. This is followed by a focus on sustainable biocuration of functional annotation, an area which has particularly felt the pressure of sequencing growth. The importance of functional annotation, how it can be submitted and the shifting role of the biocurator in the context of increasing volumes of data are all discussed. PMID:26615190

  20. Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase.

    Science.gov (United States)

    Jin, Zhinan; Johnson, Kenneth A

    2011-01-14

    A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

  1. Identification of the Structural Features of Guanine Derivatives as MGMT Inhibitors Using 3D-QSAR Modeling Combined with Molecular Docking

    Directory of Open Access Journals (Sweden)

    Guohui Sun

    2016-06-01

    Full Text Available DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT, which plays an important role in inducing drug resistance against alkylating agents that modify the O6 position of guanine in DNA, is an attractive target for anti-tumor chemotherapy. A series of MGMT inhibitors have been synthesized over the past decades to improve the chemotherapeutic effects of O6-alkylating agents. In the present study, we performed a three-dimensional quantitative structure activity relationship (3D-QSAR study on 97 guanine derivatives as MGMT inhibitors using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA methods. Three different alignment methods (ligand-based, DFT optimization-based and docking-based alignment were employed to develop reliable 3D-QSAR models. Statistical parameters derived from the models using the above three alignment methods showed that the ligand-based CoMFA (Qcv2 = 0.672 and Rncv2 = 0.997 and CoMSIA (Qcv2 = 0.703 and Rncv2 = 0.946 models were better than the other two alignment methods-based CoMFA and CoMSIA models. The two ligand-based models were further confirmed by an external test-set validation and a Y-randomization examination. The ligand-based CoMFA model (Qext2 = 0.691, Rpred2 = 0.738 and slope k = 0.91 was observed with acceptable external test-set validation values rather than the CoMSIA model (Qext2 = 0.307, Rpred2 = 0.4 and slope k = 0.719. Docking studies were carried out to predict the binding modes of the inhibitors with MGMT. The results indicated that the obtained binding interactions were consistent with the 3D contour maps. Overall, the combined results of the 3D-QSAR and the docking obtained in this study provide an insight into the understanding of the interactions between guanine derivatives and MGMT protein, which will assist in designing novel MGMT inhibitors with desired activity.

  2. Identification of the Structural Features of Guanine Derivatives as MGMT Inhibitors Using 3D-QSAR Modeling Combined with Molecular Docking.

    Science.gov (United States)

    Sun, Guohui; Fan, Tengjiao; Zhang, Na; Ren, Ting; Zhao, Lijiao; Zhong, Rugang

    2016-01-01

    DNA repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT), which plays an important role in inducing drug resistance against alkylating agents that modify the O⁶ position of guanine in DNA, is an attractive target for anti-tumor chemotherapy. A series of MGMT inhibitors have been synthesized over the past decades to improve the chemotherapeutic effects of O⁶-alkylating agents. In the present study, we performed a three-dimensional quantitative structure activity relationship (3D-QSAR) study on 97 guanine derivatives as MGMT inhibitors using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. Three different alignment methods (ligand-based, DFT optimization-based and docking-based alignment) were employed to develop reliable 3D-QSAR models. Statistical parameters derived from the models using the above three alignment methods showed that the ligand-based CoMFA (Qcv² = 0.672 and Rncv² = 0.997) and CoMSIA (Qcv² = 0.703 and Rncv² = 0.946) models were better than the other two alignment methods-based CoMFA and CoMSIA models. The two ligand-based models were further confirmed by an external test-set validation and a Y-randomization examination. The ligand-based CoMFA model (Qext² = 0.691, Rpred² = 0.738 and slope k = 0.91) was observed with acceptable external test-set validation values rather than the CoMSIA model (Qext² = 0.307, Rpred² = 0.4 and slope k = 0.719). Docking studies were carried out to predict the binding modes of the inhibitors with MGMT. The results indicated that the obtained binding interactions were consistent with the 3D contour maps. Overall, the combined results of the 3D-QSAR and the docking obtained in this study provide an insight into the understanding of the interactions between guanine derivatives and MGMT protein, which will assist in designing novel MGMT inhibitors with desired activity. PMID:27347909

  3. Extracellular nucleotide derivatives protect cardiomyctes against hypoxic stress

    DEFF Research Database (Denmark)

    Golan, O; Issan, Y; Isak, A;

    2011-01-01

    is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone...

  4. [Tabular excel editor for analysis of aligned nucleotide sequences].

    Science.gov (United States)

    Demkin, V V

    2010-01-01

    Excel platform was used for transition of results of multiple aligned nucleotide sequences obtained using the BLAST network service to the form appropriate for visual analysis and editing. Two macros operators for MS Excel 2007 were constructed. The array of aligned sequences transformed into Excel table and processed using macros operators is more appropriate for analysis than initial html data.

  5. Nucleotide excision repair I: from E.coli to yeast.

    NARCIS (Netherlands)

    J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractGenetic information is constantly deteriorating, mainly as a consequence of the action of numerous genotoxic agents. In order to cope with this fundamental problem, all living organisms have acquired a complex network of DNA repair systems to safeguard their genetic integrity. Nucleotide

  6. DNA sequence representation by trianders and determinative degree of nucleotides

    Institute of Scientific and Technical Information of China (English)

    DUPLIJ Diana; DUPLIJ Steven

    2005-01-01

    A new version of DNA walks, where nucleotides are regarded unequal in their contribution to a walk is introduced,which allows us to study thoroughly the "fine structure" of nucleotide sequences. The approach is based on the assumption that nucleotides have an inner abstract characteristic, the determinative degree, which reflects genetic code phenomenological properties and is adjusted to nucleotides physical properties. We consider each codon position independently, which gives three separate walks characterized by different angles and lengths, and that such an object is called triander which reflects the "strength"of branch. A general method for identifying DNA sequence "by triander" which can be treated as a unique "genogram" (or "gene passport") is proposed. The two- and three-dimensional trianders are considered. The difference of sequences fine structure in genes and the intergenic space is shown. A clear triplet signal in coding sequences was found which is absent in the intergenic space and is independent from the sequence length. This paper presents the topological classification oftrianders which can allow us to provide a detailed working out signatures of functionally different genomic regions.

  7. DNA Nucleotides Detection via capacitance properties of Graphene

    Science.gov (United States)

    Khadempar, Nahid; Berahman, Masoud; Yazdanpanah, Arash

    2016-05-01

    In the present paper a new method is suggested to detect the DNA nucleotides on a first-principles calculation of the electronic features of DNA bases which chemisorbed to a graphene sheet placed between two gold electrodes in a contact-channel-contact system. The capacitance properties of graphene in the channel are surveyed using non-equilibrium Green's function coupled with the Density Functional Theory. Thus, the capacitance properties of graphene are theoretically investigated in a biological environment, and, using a novel method, the effect of the chemisorbed DNA nucleotides on electrical charges on the surface of graphene is deciphered. Several parameters in this method are also extracted including Electrostatic energy, Induced density, induced electrostatic potential, Electron difference potential and Electron difference density. The qualitative and quantitative differences among these parameters can be used to identify DNA nucleotides. Some of the advantages of this approach include its ease and high accuracy. What distinguishes the current research is that it is the first experiment to investigate the capacitance properties of gaphene changes in the biological environment and the effect of chemisorbed DNA nucleotides on the surface of graphene on the charge.

  8. A Laboratory Exercise for Genotyping Two Human Single Nucleotide Polymorphisms

    Science.gov (United States)

    Fernando, James; Carlson, Bradley; LeBard, Timothy; McCarthy, Michael; Umali, Finianne; Ashton, Bryce; Rose, Ferrill F., Jr.

    2016-01-01

    The dramatic decrease in the cost of sequencing a human genome is leading to an era in which a wide range of students will benefit from having an understanding of human genetic variation. Since over 90% of sequence variation between humans is in the form of single nucleotide polymorphisms (SNPs), a laboratory exercise has been devised in order to…

  9. THE NUCLEOTIDE RECEPTORS ON MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; NELEMANS, A; VANDENAKKER, J; DENHERTOG, A

    1992-01-01

    1 The response of C2C12 mouse myotubes to stimulation with adenosine triphosphate (ATP) and other nucleotides was studied by measuring changes in membrane potential. 2 A transient hyperpolarization followed by a slowly declining depolarization of the cells was observed in the presence of ATP (10-mu-

  10. Single Nucleotide Polymorphisms Predict Symptom Severity of Autism Spectrum Disorder

    Science.gov (United States)

    Jiao, Yun; Chen, Rong; Ke, Xiaoyan; Cheng, Lu; Chu, Kangkang; Lu, Zuhong; Herskovits, Edward H.

    2012-01-01

    Autism is widely believed to be a heterogeneous disorder; diagnosis is currently based solely on clinical criteria, although genetic, as well as environmental, influences are thought to be prominent factors in the etiology of most forms of autism. Our goal is to determine whether a predictive model based on single-nucleotide polymorphisms (SNPs)…

  11. Single nucleotide polymorphisms associated with rat expressed sequences

    NARCIS (Netherlands)

    Guryev, V.; Berezikov, E.; Malik, R.; Plasterk, R.; Cuppen, E.

    2004-01-01

    Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in populations and are thus most likely to account for the majority of phenotypic and behavioral differences between individuals or strains. Although the rat is extensively studied for the latter, data on naturall

  12. Mining for Single Nucleotide Polymorphisms in Pig genome sequence data

    NARCIS (Netherlands)

    Kerstens, H.H.D.; Kollers, S.; Kommandath, A.; Rosario, del M.; Dibbits, B.W.; Kinders, S.M.; Crooijmans, R.P.M.A.; Groenen, M.A.M.

    2009-01-01

    Background - Single nucleotide polymorphisms (SNPs) are ideal genetic markers due to their high abundance and the highly automated way in which SNPs are detected and SNP assays are performed. The number of SNPs identified in the pig thus far is still limited. Results - A total of 4.8 million whole g

  13. Pinched flow fractionation devices for detection of single nucleotide polymorphisms

    DEFF Research Database (Denmark)

    Larsen, A.V.; Poulsen, L.; Birgens, H.;

    2008-01-01

    We demonstrate a new and flexible micro fluidic based method for genotyping single nucleotide polymorphisms ( SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a micro fluidic pinched flow fractionation (PFF) device. The micro fluidic PFF devices wi...

  14. The nucleotide sequences of two leghemoglobin genes from soybean

    DEFF Research Database (Denmark)

    Wiborg, O; Hyldig-Nielsen, J J; Jensen, E O;

    1982-01-01

    We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences in identical positions. Comparison of the coding sequences with known amino-acid sequences of soybean leghemoglobins suggest that the two genes corresp...

  15. Synthesis, bioanalysis and pharmacology of nucleoside and nucleotide analogs

    NARCIS (Netherlands)

    Jansen, R.S.

    2009-01-01

    Nucleoside analogs are an important class of drugs in anticancer and antiviral therapy. The compounds are, however, only active after intracellular conversion to their mono-, di- and triphosphate nucleotide form. In this thesis the development of sensitive liquid chromatography coupled to tandem mas

  16. Global regulation of nucleotide biosynthetic genes by c-Myc.

    Directory of Open Access Journals (Sweden)

    Yen-Chun Liu

    Full Text Available BACKGROUND: The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2 on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.

  17. Quantum processes in 8-Oxo-Guanine-Ru(bipyridine){3/2+} photosynthetic systems of artificial minimal cells

    Science.gov (United States)

    Tamulis, Arvydas; Grigalavicius, Mantas; Krisciukaitis, Sarunas; Medzevicius, Giedrius

    2011-06-01

    Density functional theory methods were used to investigate various self-assembled photoactive bioorganic systems of interest for artificial minimal cells. The cell systems studied are based on nucleotides or their compounds and consisted of up to 123 atoms (not including the associated water or methanol solvent shells) and are up to 2.5 nm in diameter. The electron correlation interactions responsible for the weak hydrogen and Van derWaals chemical bonds increase due to the addition of a polar solvent (water or methanol). The precursor fatty acid molecules of the system also play a critical role in the quantum mechanical interaction based self-assembly of the photosynthetic center and the functioning of the photosynthetic processes of the artificial minimal cells. The distances between the separated sensitizer, fatty acid precursor, and methanol molecules are comparable to Van derWaals and hydrogen bonding radii. As a result the associated electron correlation interactions compress the overall system, resulting in an even smaller gap between the highest occupied molecular orbital (HOMO), and lowest unoccupied molecular orbital (LUMO) electron energy levels and photoexcited electron tunnelling occurs from the sensitizer (either Ru(bpy){3/2+} or [Ru(bpy)2(4-Bu-4'-Me-2,2'-bpy)]2++ derivatives) to the precursor fatty acid molecules (notation used: Me = methyl; Bu = butyl; bpy = bipyridine). The shift of the absorption spectrum to the red for the artificial protocell photosynthetic centers might be considered as the measure of the complexity of these systems.

  18. Reactivities of radicals of adenine and guanine towards reactive oxygen species and reactive nitrogen oxide species: OH rad and NO 2rad

    Science.gov (United States)

    Agnihotri, Neha; Mishra, P. C.

    2011-02-01

    Reactions of radicals of the DNA bases with reactive oxygen species and reactive nitrogen oxide species produce mutagenic products. We have studied reactivities of all the carbon sites of radicals of adenine A(-H) rad and guanine G(-H) rad obtained by removal of H-atoms from their nitrogen sites towards OH rad and NO 2rad . We studied stabilities of A(-H) rad and G(-H) rad and binding energies of their adducts with each of OH rad and NO 2rad using density functional theoretic and MP2 calculations employing the AUG-cc-pVDZ basis set. Solvation in aqueous media was treated using the polarization continuum model. The results obtained explain experimental observations.

  19. N,N,O and N,O,N Meridional cis Coordination of Two Guanines to Copper(II) by d(CGCGCG)2.

    Science.gov (United States)

    Rohner, Melanie; Medina-Molner, Alfredo; Spingler, Bernhard

    2016-06-20

    Many research groups study the generation of supramolecular n-dimensional arrays by combining metals with DNA building blocks. Most of the time, the natural nucleobases are modified to obtain higher-affinity metal binding sites. Using unmodified nucleobases avoids a potentially difficult synthesis; however, they have the possible disadvantage of a less defined and/or weaker coordination mode of the metal. Structural studies on the behavior of copper(II) as a linking metal and guanine as the natural ligand for metals in unmodified DNA are reported. Previously, the ability of mono- and dinuclear metal complexes to induce Z-DNA has been explored [Medina-Molner, A.; Spingler, B. Chem. Commun. 2012, 48, 1961; Medina-Molner, A.; Rohner, M.; Pandiarajan, D.; Spingler, B. Dalton Trans. 2015, 44, 3664]. Herein, X-ray crystallographic studies of the structures resulting from the combination of copper(II) ions with DNA hexamers of the general sequence d(CG)3 are presented. Three different packing motifs were observed in three crystal structures with resolutions ranging from 2.15 to 1.45 Å. The motifs are dependent upon other cations being present and/or the crystallization conditions. The first examples of intramolecular O6,N7-chelates of a neutral purine nucleobase to copper(II) were obtained as well as the first meridional N,N,O and N,O,N coordination modes of two guanines to copper(II). The fascinating coordination chemistry of copper(II) complexes generated by the Z-DNA oligonucleotides and the differences to simple nucleobases complexes with copper(II) are discussed in detail. PMID:27266259

  20. N,N,O and N,O,N Meridional cis Coordination of Two Guanines to Copper(II) by d(CGCGCG)2.

    Science.gov (United States)

    Rohner, Melanie; Medina-Molner, Alfredo; Spingler, Bernhard

    2016-06-20

    Many research groups study the generation of supramolecular n-dimensional arrays by combining metals with DNA building blocks. Most of the time, the natural nucleobases are modified to obtain higher-affinity metal binding sites. Using unmodified nucleobases avoids a potentially difficult synthesis; however, they have the possible disadvantage of a less defined and/or weaker coordination mode of the metal. Structural studies on the behavior of copper(II) as a linking metal and guanine as the natural ligand for metals in unmodified DNA are reported. Previously, the ability of mono- and dinuclear metal complexes to induce Z-DNA has been explored [Medina-Molner, A.; Spingler, B. Chem. Commun. 2012, 48, 1961; Medina-Molner, A.; Rohner, M.; Pandiarajan, D.; Spingler, B. Dalton Trans. 2015, 44, 3664]. Herein, X-ray crystallographic studies of the structures resulting from the combination of copper(II) ions with DNA hexamers of the general sequence d(CG)3 are presented. Three different packing motifs were observed in three crystal structures with resolutions ranging from 2.15 to 1.45 Å. The motifs are dependent upon other cations being present and/or the crystallization conditions. The first examples of intramolecular O6,N7-chelates of a neutral purine nucleobase to copper(II) were obtained as well as the first meridional N,N,O and N,O,N coordination modes of two guanines to copper(II). The fascinating coordination chemistry of copper(II) complexes generated by the Z-DNA oligonucleotides and the differences to simple nucleobases complexes with copper(II) are discussed in detail.

  1. High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation.

    Science.gov (United States)

    Villiger, Thomas K; Steinhoff, Robert F; Ivarsson, Marija; Solacroup, Thomas; Stettler, Matthieu; Broly, Hervé; Krismer, Jasmin; Pabst, Martin; Zenobi, Renato; Morbidelli, Massimo; Soos, Miroslav

    2016-07-10

    Recent advances in miniaturized cell culture systems have facilitated the screening of media additives on productivity and protein quality attributes of mammalian cell cultures. However, intracellular components are not routinely measured due to the limited throughput of available analytical techniques. In this work, time profiling of intracellular nucleotides and nucleotide sugars of CHO-S cell fed-batch processes in a micro-scale bioreactor system was carried out using a recently developed high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). Supplementation of various media additives significantly altered the intracellular nucleotides and nucleotide sugars that are inextricably linked to the process of glycosylation. The results revealed that UDP-Gal synthesis appeared to be particularly limiting whereas the impact of elevated UDP-GlcNAc and GDP-Fuc levels on the final glycosylation patterns was only marginally important. In contrast, manganese and asparagine supplementation altered the glycan profiles without affecting intracellular components. The combination of miniaturized cell cultures and high-throughput analytical techniques serves therefore as a useful tool for future quality driven media optimization studies. PMID:27131894

  2. Exonuclease activity and P nucleotide addition in the generation of the expressed immunoglobulin repertoire

    Directory of Open Access Journals (Sweden)

    Sewell William

    2004-09-01

    Full Text Available Abstract Background Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides. Results A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p Conclusions The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences.

  3. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.

    2013-09-03

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin-binding domain. Despite their functional similarities, the plant CNGC family members appear to have different conserved amino acid motifs within corresponding functional domains than animal and bacterial CNGCs do. Here we describe the development and application of methods employing plant CNGC-specific sequence motifs as diagnostic tools to identify novel candidate channels in different plants. These methods are used to evaluate the validity of annotations of putative orthologs of CNGCs from plant genomes. The methods detail how to employ regular expressions of conserved amino acids in functional domains of annotated CNGCs and together with Web tools such as PHI-BLAST and ScanProsite to identify novel candidate CNGCs in species including Physcomitrella patens. © Springer Science+Business Media New York 2013.

  4. The nucleotide exchange factors of Hsp70 molecular chaperone

    Directory of Open Access Journals (Sweden)

    Andreas eBracher

    2015-04-01

    Full Text Available Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1 and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell.

  5. Isolation and nucleotide sequence of the gene encoding human rhodopsin.

    OpenAIRE

    Nathans, J; Hogness, D S

    1984-01-01

    We have isolated and completely sequenced the gene encoding human rhodopsin. The coding region of the human rhodopsin gene is interrupted by four introns, which are located at positions analogous to those found in the previously characterized bovine rhodopsin gene. The amino acid sequence of human rhodopsin, deduced from the nucleotide sequence of its gene, is 348 residues long and is 93.4% homologous to that of bovine rhodopsin. Interestingly, those portions of the polypeptide chain predicte...

  6. Nucleotide Sequence - KOME | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ..._db.zip File URL: ftp://ftp.biosciencedbc.jp/archive/kome/LATEST/kome_ine_full_se...quence_db.zip File size: 19 MB File name: FASTA: kome_ine_full_sequence_db.fasta.zip File URL: ftp://ftp.biosciencedbc.jp/archiv...rtio About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Nucleotide Sequence - KOME | LSDB Archive ...

  7. MGMT expression: insights into its regulation. 2. Single nucleotide polymorphisms

    OpenAIRE

    Iatsyshyna A. P.; Pidpala O. V.; Lukash L. L.

    2013-01-01

    High intra- and interindividual variations in the expression levels of the human O6-methylguanine-DNA methyltransferase (MGMT) gene have been observed. This DNA repair enzyme can be a cause of resistance of cancer cells to alkylating chemotherapy. It has been studied the association of single nucleotide polymorphisms (SNPs) of MGMT with the risk for different types of cancer, progression-free survival in patients with cancer treated with alkylating chemotherapy, as well as an effect of SNPs o...

  8. Utilizing Single Nucleotide Polymorphism Analysis in Determining Parentage of Cattle

    OpenAIRE

    Elbert, Nicole M.

    2013-01-01

    Parentage identification within cattle herds is an important aspect of record keeping. It is essential for accurate registration within a purebred association and decision making for production purposes, such as replacement heifer and sire selection. Methods used to identify parentage have evolved from utilizing blood protein antigens, restriction fragment length polymorphism (RFLP) and microsatellites to the current technology of analyzing DNA profiles for differing single nucleotide polymor...

  9. The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Lykke, Anne; Rasmussen, Lene Juel

    2010-01-01

    Several enzymes of the metabolic pathways responsible for metabolism of cytosolic ribonucleotides and deoxyribonucleotides are located in mitochondria. Studies described in this paper suggest dysfunction of the mitochondria to affect these metabolic pathways and limit the available levels of...... cytosolic ribonucleotides and deoxyribonucleotides, which in turn can result in aberrant RNA and DNA synthesis. Mitochondrial dysfunction has been linked to genomic instability, and it is possible that the limiting effect of mitochondrial dysfunction on the levels of nucleotides and resulting aberrant RNA...

  10. Single nucleotide polymorphisms of complement component 5 and periodontitis

    OpenAIRE

    L. Chai; Zee, KY; Song, YQ; Leung, WK

    2010-01-01

    BACKGROUND AND OBJECTIVE: Polymorphisms of host defence genes might increase one's risks for periodontitis. This study investigated whether tagging single nucleotide polymorphisms (SNPs) of the gene encoding complement component 5 (C5) are associated with periodontitis in a Hong Kong Chinese population. MATERIAL AND METHODS: Eleven tagging SNPs of 229 patients with at least moderate periodontitis and 207 control subjects without periodontitis were genotyped using an i-plexGOLD MassARRAY mass-...

  11. Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

    Energy Technology Data Exchange (ETDEWEB)

    Lo, A.; Yang, H.L.

    1990-02-13

    This patent describes a composition of matter that is specific for {ital Neisseria gonorrhoeae}. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria gonorrhoeae} to the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria meningitidis} is greater than about five. The ratio being obtained by a method described.

  12. Mitochondria as determinant of nucleotide pools and chromosomal stability

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Munch-Petersen, Birgitte; Stevnsner, Tinna;

    2007-01-01

    Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochon...... mitochondrial activity. Our results suggest that mitochondria are central players in maintaining genomic stability and in controlling essential nuclear processes such as upholding a balanced supply of nucleotides....

  13. Analysis of Single Nucleotide Polymorphism Panels for Bovine DNA Identification

    OpenAIRE

    Blanchard, Kimberly A.

    2013-01-01

    Single nucleotide polymorphisms (SNPs) are single base-pair variations that exist between individuals. There are approximately a million or more SNPs located throughout the genome of each individual animal. Therefore, by taking advantage of these unique polymorphisms, SNPs can be used to resolve questions of unknown parentage in the livestock industry. Currently a panel of 88 SNPs, obtained from a panel of 121 SNPs originally created by USDA-MARC, is commercially available from Fluidigm®. The...

  14. Nucleotide Sequencing and Identification of Some Wild Mushrooms

    Directory of Open Access Journals (Sweden)

    Sudip Kumar Das

    2013-01-01

    Full Text Available The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers fragment of the genomic DNA of 8 wild edible mushrooms (collected from Eastern Chota Nagpur Plateau of West Bengal, India was amplified using ITS1 (Internal Transcribed Spacers 1 and ITS2 primers and subjected to nucleotide sequence determination for identification of mushrooms as mentioned. The sequences were aligned using ClustalW software program. The aligned sequences revealed identity (homology percentage from GenBank data base of Amanita hemibapha [CN (Chota Nagpur 1, % identity 99 (JX844716.1], Amanita sp. [CN 2, % identity 98 (JX844763.1], Astraeus hygrometricus [CN 3, % identity 87 (FJ536664.1], Termitomyces sp. [CN 4, % identity 90 (JF746992.1], Termitomyces sp. [CN 5, % identity 99 (GU001667.1], T. microcarpus [CN 6, % identity 82 (EF421077.1], Termitomyces sp. [CN 7, % identity 76 (JF746993.1], and Volvariella volvacea [CN 8, % identity 100 (JN086680.1]. Although out of 8 mushrooms 4 could be identified up to species level, the nucleotide sequences of the rest may be relevant to further characterization. A phylogenetic tree is constructed using Neighbor-Joining method showing interrelationship between/among the mushrooms. The determined nucleotide sequences of the mushrooms may provide additional information enriching GenBank database aiding to molecular taxonomy and facilitating its domestication and characterization for human benefits.

  15. Genome-wide patterns of nucleotide polymorphism in domesticated rice.

    Directory of Open Access Journals (Sweden)

    Ana L Caicedo

    2007-09-01

    Full Text Available Domesticated Asian rice (Oryza sativa is one of the oldest domesticated crop species in the world, having fed more people than any other plant in human history. We report the patterns of DNA sequence variation in rice and its wild ancestor, O. rufipogon, across 111 randomly chosen gene fragments, and use these to infer the evolutionary dynamics that led to the origins of rice. There is a genome-wide excess of high-frequency derived single nucleotide polymorphisms (SNPs in O. sativa varieties, a pattern that has not been reported for other crop species. We developed several alternative models to explain contemporary patterns of polymorphisms in rice, including a (i selectively neutral population bottleneck model, (ii bottleneck plus migration model, (iii multiple selective sweeps model, and (iv bottleneck plus selective sweeps model. We find that a simple bottleneck model, which has been the dominant demographic model for domesticated species, cannot explain the derived nucleotide polymorphism site frequency spectrum in rice. Instead, a bottleneck model that incorporates selective sweeps, or a more complex demographic model that includes subdivision and gene flow, are more plausible explanations for patterns of variation in domesticated rice varieties. If selective sweeps are indeed the explanation for the observed nucleotide data of domesticated rice, it suggests that strong selection can leave its imprint on genome-wide polymorphism patterns, contrary to expectations that selection results only in a local signature of variation.

  16. Single Nucleotide Polymorphisms and Linkage Disequilibrium in Sunflower

    Science.gov (United States)

    Kolkman, Judith M.; Berry, Simon T.; Leon, Alberto J.; Slabaugh, Mary B.; Tang, Shunxue; Gao, Wenxiang; Shintani, David K.; Burke, John M.; Knapp, Steven J.

    2007-01-01

    Genetic diversity in modern sunflower (Helianthus annuus L.) cultivars (elite oilseed inbred lines) has been shaped by domestication and breeding bottlenecks and wild and exotic allele introgression−the former narrowing and the latter broadening genetic diversity. To assess single nucleotide polymorphism (SNP) frequencies, nucleotide diversity, and linkage disequilibrium (LD) in modern cultivars, alleles were resequenced from 81 genic loci distributed throughout the sunflower genome. DNA polymorphisms were abundant; 1078 SNPs (1/45.7 bp) and 178 insertions-deletions (INDELs) (1/277.0 bp) were identified in 49.4 kbp of DNA/genotype. SNPs were twofold more frequent in noncoding (1/32.1 bp) than coding (1/62.8 bp) sequences. Nucleotide diversity was only slightly lower in inbred lines (θ = 0.0094) than wild populations (θ = 0.0128). Mean haplotype diversity was 0.74. When extraploted across the genome (∼3500 Mbp), sunflower was predicted to harbor at least 76.4 million common SNPs among modern cultivar alleles. LD decayed more slowly in inbred lines than wild populations (mean LD declined to 0.32 by 5.5 kbp in the former, the maximum physical distance surveyed), a difference attributed to domestication and breeding bottlenecks. SNP frequencies and LD decay are sufficient in modern sunflower cultivars for very high-density genetic mapping and high-resolution association mapping. PMID:17660563

  17. Electrochemical Quantification of Single Nucleotide Polymorphisms Using Nanoparticle Probes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guodong; Lin, Yuehe

    2007-08-29

    We report a new approach for electrochemical quantification of single-nucleotide polymorphisms (SNPs) using nanoparticle probes. The principle is based on DNA polymerase I (klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the Watson-Crick base pairing rule. After liquid hybridization events occurred among biotinylated DNA probes, mutant DNA, and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a biotin-avidin affinity reaction and magnetic separation. A cadmium phosphate-loaded apoferritin nanoparticle probe, which is modified with nucleotides and is complementary to the mutant site, is coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Subsequent electrochemical stripping analysis of the cadmium component of coupled nanoparticle probes provides a means to quantify the concentration of mutant DNA. The method is sensitive enough to detect 21.5 attomol mutant DNA, which will enable the quantitative analysis of nucleic acid without polymerase chain reaction pre-amplification. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to accurately determine SNPs with frequencies as low 0.01. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection.

  18. The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Fajardo, Alexandra M.; Piazza, Gary A. [Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Suite 3029, Mobile, AL 36604 (United States); Tinsley, Heather N., E-mail: htinsley@montevallo.edu [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)

    2014-02-26

    For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

  19. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water.

    Science.gov (United States)

    Cafferty, Brian J; Fialho, David M; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V

    2016-04-25

    The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life.

  20. Effects of bovine cytochrome P450 single-nucleotide polymorphism, forage type and body condition on production traits in cattle.

    Science.gov (United States)

    Sales, M A; Larson, M J; Reiter, S T; Brown, A H; Brown, M A; Looper, M L; Coffey, K P; Rosenkrans, C F

    2012-08-01

    Relating single-nucleotide polymorphisms (SNP) to cows with acceptable productivity could benefit cattle breeders in areas where tall fescue is the predominant forage. This study aimed to (i) identify SNPs in bovine cytochrome P450 3A28 (CYP3A28) and (ii) determine the associations between SNP genotype, forage and cow body condition (BC). Genotype (CC, CG or GG) and forage [Kentucky-31 wild-type endophyte-infected tall fescue (KY+) vs. bermudagrass] effects on milk volume and quality were determined in Herd 1 cows (123 cows); in Herd 2 (99 cows), genotype and BC (low vs. moderate) effects on ovarian follicle size, calving date and calving per cent were determined; and in Herd 3 (114 cows), effects of genotype and fescue cultivar [KY+ vs. non-toxic endophyte-infected tall fescue (HiMag4)] were related to calving per cent, calving date and weaning weights of both cow and her calf. A cytosine (C) to guanine (G) transversion at base 994 (C994G) in CYP3A28 was identified. There was a genotype × forage type interaction (p milk protein in Herd 1 cows; CC cows grazing bermudagrass had greater milk protein percentage in relation to other cows in the herd. In Herd 2, BC and genotype × BC tended (p < 0.10) to influence follicle size and Julian calving date respectively. Diameter of the largest follicle tended to be larger in moderate BC than in low-BC cows; whereas, CC and CG cows in moderate BC and homozygous (CC and GG) cows in low BC tended to calve 14 days earlier in relation to CG cows in low BC. In Herd 3, there was a genotype × forage type interaction (p < 0.05) on calving per cent, Julian calving date and calf weaning weight. In this study, genetic alterations (G allele at C994G) coupled with nutritional factors (low BC and toxic tall fescue) resulted in overall lower productivity in cows.

  1. A Fluorimetric Readout Reporting the Kinetics of Nucleotide-Induced Human Ribonucleotide Reductase Oligomerization

    OpenAIRE

    Fu, Yuan; Lin, Hongyu; Wisitpitthaya, Somsinee; Blessing, William A.; Aye, Yimon

    2014-01-01

    Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typicall...

  2. Performance-enhancing effects of dietary nucleotides: do mitochondria play a role?

    Directory of Open Access Journals (Sweden)

    Sergej M. Ostojic

    2015-09-01

    Full Text Available Nucleotides are group of natural biomonomeric molecules and novel dietary supplements with performance-enhancing attributes. However, their mechanisms of action and target biological structures are poorly understood and identified. This short paper overviews the possible role of mitochondria during the utilization of nucleotides for exercise performance. Mitochondria-related effects of nucleotides have been identified, along with obstacles for dietary nucleotides delivery to the organelle.

  3. n-Nucleotide circular codes in graph theory.

    Science.gov (United States)

    Fimmel, Elena; Michel, Christian J; Strüngmann, Lutz

    2016-03-13

    The circular code theory proposes that genes are constituted of two trinucleotide codes: the classical genetic code with 61 trinucleotides for coding the 20 amino acids (except the three stop codons {TAA,TAG,TGA}) and a circular code based on 20 trinucleotides for retrieving, maintaining and synchronizing the reading frame. It relies on two main results: the identification of a maximal C(3) self-complementary trinucleotide circular code X in genes of bacteria, eukaryotes, plasmids and viruses (Michel 2015 J. Theor. Biol. 380, 156-177. (doi:10.1016/j.jtbi.2015.04.009); Arquès & Michel 1996 J. Theor. Biol. 182, 45-58. (doi:10.1006/jtbi.1996.0142)) and the finding of X circular code motifs in tRNAs and rRNAs, in particular in the ribosome decoding centre (Michel 2012 Comput. Biol. Chem. 37, 24-37. (doi:10.1016/j.compbiolchem.2011.10.002); El Soufi & Michel 2014 Comput. Biol. Chem. 52, 9-17. (doi:10.1016/j.compbiolchem.2014.08.001)). The univerally conserved nucleotides A1492 and A1493 and the conserved nucleotide G530 are included in X circular code motifs. Recently, dinucleotide circular codes were also investigated (Michel & Pirillo 2013 ISRN Biomath. 2013, 538631. (doi:10.1155/2013/538631); Fimmel et al. 2015 J. Theor. Biol. 386, 159-165. (doi:10.1016/j.jtbi.2015.08.034)). As the genetic motifs of different lengths are ubiquitous in genes and genomes, we introduce a new approach based on graph theory to study in full generality n-nucleotide circular codes X, i.e. of length 2 (dinucleotide), 3 (trinucleotide), 4 (tetranucleotide), etc. Indeed, we prove that an n-nucleotide code X is circular if and only if the corresponding graph [Formula: see text] is acyclic. Moreover, the maximal length of a path in [Formula: see text] corresponds to the window of nucleotides in a sequence for detecting the correct reading frame. Finally, the graph theory of tournaments is applied to the study of dinucleotide circular codes. It has full equivalence between the combinatorics

  4. The nucleotide sequence and genome organization of Plasmopara halstedii virus

    Directory of Open Access Journals (Sweden)

    Göpfert Jens C

    2011-03-01

    Full Text Available Abstract Background Only very few viruses of Oomycetes have been studied in detail. Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. However, complete nucleotide sequences and data on the genome organization were lacking. Methods Viral RNA of different P. halstedii isolates was subjected to nucleotide sequencing and analysis of the viral genome. The N-terminal sequence of the viral coat protein was determined using Top-Down MALDI-TOF analysis. Results The complete nucleotide sequences of both single-stranded RNA segments (RNA1 and RNA2 were established. RNA1 consisted of 2793 nucleotides (nt exclusive its 3' poly(A tract and a single open-reading frame (ORF1 of 2745 nt. ORF1 was framed by a 5' untranslated region (5' UTR of 18 nt and a 3' untranslated region (3' UTR of 30 nt. ORF1 contained motifs of RNA-dependent RNA polymerases (RdRp and showed similarities to RdRp of Scleropthora macrospora virus A (SmV A and viruses within the Nodaviridae family. RNA2 consisted of 1526 nt exclusive its 3' poly(A tract and a second ORF (ORF2 of 1128 nt. ORF2 coded for the single viral coat protein (CP and was framed by a 5' UTR of 164 nt and a 3' UTR of 234 nt. The deduced amino acid sequence of ORF2 was verified by nano-LC-ESI-MS/MS experiments. Top-Down MALDI-TOF analysis revealed the N-terminal sequence of the CP. The N-terminal sequence represented a region within ORF2 suggesting a proteolytic processing of the CP in vivo. The CP showed similarities to CP of SmV A and viruses within the Tombusviridae family. Fragments of RNA1 (ca. 1.9 kb and RNA2 (ca. 1.4 kb were used to analyze the nucleotide sequence variation of virions in different P. halstedii isolates. Viral sequence variation was 0.3% or less regardless of their host's pathotypes, the geographical origin and the sensitivity towards the fungicide metalaxyl. Conclusions The results showed the presence of a single and new

  5. Nucleotide sequences specific to Brucella and methods for the detection of Brucella

    Science.gov (United States)

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.

    2009-02-24

    Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  6. AVP-stimulated nucleotide secretion in perfused mouse medullary thick ascending limb and cortical collecting duct

    DEFF Research Database (Denmark)

    Odgaard, Elvin V. P.; Prætorius, Helle; Leipziger, Jens Georg

    2009-01-01

    is stimulated remain elusive. Here, we investigate the phenomenon of nucleotide secretion in intact, perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor adapted to register nucleotides in the tubular outflow...

  7. New water-soluble ruthenium(II) terpyridine complexes for anticancer activity: synthesis, characterization, activation kinetics, and interaction with guanine derivatives.

    Science.gov (United States)

    Rilak, Ana; Bratsos, Ioannis; Zangrando, Ennio; Kljun, Jakob; Turel, Iztok; Bugarčić, Živadin D; Alessio, Enzo

    2014-06-16

    With the aim of assessing whether ruthenium(II) compounds with meridional geometry might be utilized as potential antitumor agents, a series of new, water-soluble, monofunctional ruthenium(II) complexes of the general formula mer-[Ru(L3)(N-N)X][Y]n (where L3 = 2,2':6',2″-terpyridine (tpy) or 4'-chloro-2,2':6',2″-terpyridine (Cl-tpy), N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach), or 2,2'-bipyridine (bpy); X = Cl or dmso-S; Y = Cl, PF6, or CF3SO3; n = 1 or 2, depending on the nature of X) were synthesized. All complexes were fully characterized by elemental analysis and spectroscopic techniques (IR, UV/visible, and 1D and 2D NMR), and for three of them, i.e., [Ru(Cl-tpy)(bpy)Cl][Cl] (3Cl), [Ru(Cl-tpy)(en)(dmso-S)][Y]2 [Y = PF6 (6PF6), CF3SO3 (6OTf)] and [Ru(Cl-tpy)(bpy)(dmso-S)][CF3SO3]2 (8OTf), the X-ray structure was also determined. The new terpyridine complexes, with the exception of 8, are well soluble in water (>25 mg/mL). (1)H and (31)P NMR spectroscopy studies performed on the three selected complexes [Ru(Cl-tpy)(N-N)Cl](+) [N-N = en (1), dach (2), and bpy (3)] demonstrated that, after hydrolysis of the Cl ligand, they are capable of interacting with guanine derivatives [i.e., 9-methylguanine (9MeG) or guanosine-5'-monophosphate (5'-GMP)] through N7, forming monofunctional adducts with rates and extents that depend strongly on the nature of N-N: 1 ≈ 2 ≫ 3. In addition, compound 1 shows high selectivity toward 5'-GMP compared to adenosine-5'-monophosphate (5'-AMP), in a competition experiment. Quantitative kinetic investigations on 1 and 2 were performed by means of UV/visible spectroscopy. Overall, the complexes with bidentate aliphatic diamines proved to be superior to those with bpy in terms of solubility and reactivity (i.e., release of Cl(-) and capability to bind guanine derivatives). Contrary to the chlorido compounds, the corresponding dmso derivatives proved to be inert (viz., they do not release the monodentate ligand) in

  8. Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.

    Science.gov (United States)

    Cervoni, L; Lascu, I; Xu, Y; Gonin, P; Morr, M; Merouani, M; Janin, J; Giartosio, A

    2001-04-17

    The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications. PMID:11294625

  9. Bioinformatics comparison of sulfate-reducing metabolism nucleotide sequences

    Science.gov (United States)

    Tremberger, G.; Dehipawala, Sunil; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    The sulfate-reducing bacteria can be traced back to 3.5 billion years ago. The thermodynamics details of the sulfur cycle have been well documented. A recent sulfate-reducing bacteria report (Robator, Jungbluth, et al , 2015 Jan, Front. Microbiol) with Genbank nucleotide data has been analyzed in terms of the sulfite reductase (dsrAB) via fractal dimension and entropy values. Comparison to oil field sulfate-reducing sequences was included. The AUCG translational mass fractal dimension versus ATCG transcriptional mass fractal dimension for the low temperature dsrB and dsrA sequences reported in Reference Thirteen shows correlation R-sq ~ 0.79 , with a probably of about 3% in simulation. A recent report of using Cystathionine gamma-lyase sequence to produce CdS quantum dot in a biological method, where the sulfur is reduced just like in the H2S production process, was included for comparison. The AUCG mass fractal dimension versus ATCG mass fractal dimension for the Cystathionine gamma-lyase sequences was found to have R-sq of 0.72, similar to the low temperature dissimilatory sulfite reductase dsr group with 3% probability, in contrary to the oil field group having R-sq ~ 0.94, a high probable outcome in the simulation. The other two simulation histograms, namely, fractal dimension versus entropy R-sq outcome values, and di-nucleotide entropy versus mono-nucleotide entropy R-sq outcome values are also discussed in the data analysis focusing on low probability outcomes.

  10. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    Science.gov (United States)

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016.

  11. Dietary nucleotides influence immune responses and intestinal morphology of red drum Sciaenops ocellatus.

    Science.gov (United States)

    Cheng, Zhenyan; Buentello, Alejandro; Gatlin, Delbert M

    2011-01-01

    Dietary nucleotides have been shown to benefit many physiological and nutritional functions in higher vertebrates and fish. Therefore, a 6-week feeding trial was conducted to evaluate the effects of graded levels of a commercial nucleotide product on growth performance, immune responses and intestinal morphology of juvenile red drum (initial average weight of 7.1g). The basal diet was formulated to contain 40% protein, 10% lipid and a digestible energy level of 3.5 kcal g(-1). Two levels of nucleotide (Ascogen P(®), 0.5% and 1% of diet) were added to the basal diet with menhaden fishmeal and menhaden oil adjusted to provide isonitrogenous and isolipidic diets. Nucleotide supplementation tended to improve weight gain and survival of red drum, but not at a significant level. Neutrophil oxidative radical anion production and serum lysozyme activity tended to be higher for fish fed diets supplemented with nucleotide, while extracellular superoxide anion production of head kidney macrophages from fish fed diets with 1% nucleotide was significantly (Pfish fed 0.5% nucleotide diet and the basal diet. Nucleotide supplementation significantly (Pfish fed with diets supplemented with nucleotides. It is therefore possible to use dietary nucleotides supplementation to significantly enhance the intestinal structure of red drum. Likewise, nucleotides in the diet may improve some components of the non-specific immune response of this sciaenid fish.

  12. Nucleotide sequence of a spinach chloroplast valine tRNA.

    OpenAIRE

    Sprouse, H M; Kashdan, M; Otis, L; Dudock, B

    1981-01-01

    The nucleotide sequence of a spinach chloroplast valine tRNA (sp. chl. tRNA Val) has been determined. This tRNA shows essentially equal homology to prokaryotic valine tRNAs (58-65% homology) and to the mitochondrial valine tRNAs of lower eukaryotes (yeast and N. crassa, 61-62% homology). Sp. chl. tRNA Val shows distinctly lower homology to mouse mitochondrial valine tRNA (53% homology) and to eukaryotic cytoplasmic valine tRNAs (47-53% homology). Sp. chl. tRNA Val, like all other chloroplast ...

  13. The complete nucleotide sequence of pelargonium leaf curl virus.

    Science.gov (United States)

    McGavin, Wendy J; MacFarlane, Stuart A

    2016-05-01

    Investigation of a tombusvirus isolated from tulip plants in Scotland revealed that it was pelargonium leaf curl virus (PLCV) rather than the originally suggested tomato bushy stunt virus. The complete sequence of the PLCV genome was determined for the first time, revealing it to be 4789 nucleotides in size and to have an organization similar to that of the other, previously described tombusviruses. Primers derived from the sequence were used to construct a full-length infectious clone of PLCV that recapitulates the disease symptoms of leaf curling in systemically infected pelargonium plants. PMID:26906694

  14. Metal-based chemosensors for amino acids, peptides, and nucleotides

    OpenAIRE

    Buryak, Andrey

    2007-01-01

    An organometallic 4d transition metal complex [Cp*RhCl2]2, together with commercially available dyes, was used to construct indicator displacement assays (IDAs) for the detection of peptides, amino acids, and nucleotides. The combination of the Cp*Rh complex with the dye azophloxine was found to form a chemosensing ensemble for the sequence-selective detection of histidine- and methionine-containing peptides in water at neutral pH. A strong interaction of the rhodium complex with peptides bea...

  15. Metal-based chemosensors for amino acids, peptides, and nucleotides

    OpenAIRE

    Buryak, Andrey; Severin, Kay

    2008-01-01

    An organometallic 4d transition metal complex [Cp*RhCl2]2, together with commercially available dyes, was used to construct indicator displacement assays (IDAs) for the detection of peptides, amino acids, and nucleotides. The combination of the Cp*Rh complex with the dye azophloxine was found to form a chemosensing ensemble for the sequence-selective detection of histidine- and methionine-containing peptides in water at neutral pH. A strong interaction of the rhodium complex with peptides bea...

  16. The complete nucleotide sequence of pelargonium leaf curl virus.

    Science.gov (United States)

    McGavin, Wendy J; MacFarlane, Stuart A

    2016-05-01

    Investigation of a tombusvirus isolated from tulip plants in Scotland revealed that it was pelargonium leaf curl virus (PLCV) rather than the originally suggested tomato bushy stunt virus. The complete sequence of the PLCV genome was determined for the first time, revealing it to be 4789 nucleotides in size and to have an organization similar to that of the other, previously described tombusviruses. Primers derived from the sequence were used to construct a full-length infectious clone of PLCV that recapitulates the disease symptoms of leaf curling in systemically infected pelargonium plants.

  17. Patterns of nucleotides that flank substitutions in human orthologous genes

    Directory of Open Access Journals (Sweden)

    Huang Zhuoran

    2010-07-01

    Full Text Available Abstract Background Sequence context is an important aspect of base mutagenesis, and three-base periodicity is an intrinsic property of coding sequences. However, how three-base periodicity is influenced in the vicinity of substitutions is still unclear. The effect of context on mutagenesis should be revealed in the usage of nucleotides that flank substitutions. Relative entropy (also known as Kullback-Leibler divergence is useful for finding unusual patterns in biological sequences. Results Using relative entropy, we visualized the periodic patterns in the context of substitutions in human orthologous genes. Neighbouring patterns differed both among substitution categories and within a category that occurred at three codon positions. Transition tended to occur in periodic sequences relative to transversion. Periodic signals were stronger in a set of flanking sequences of substitutions that occurred at the third-codon positions than in those that occurred at the first- or second-codon positions. To determine how the three-base periodicity was affected near the substitution sites, we fitted a sine model to the values of the relative entropy. A sine of period equal to 3 is a good approximation for the three-base periodicity at sites not in close vicinity to some substitutions. These periods were interrupted near the substitution site and then reappeared away from substitutions. A comparative analysis between the native and codon-shuffled datasets suggested that the codon usage frequency was not the sole origin of the three-base periodicity, implying that the native order of codons also played an important role in this periodicity. Synonymous codon shuffling revealed that synonymous codon usage bias was one of the factors responsible for the observed three-base periodicity. Conclusions Our results offer an efficient way to illustrate unusual periodic patterns in the context of substitutions and provide further insight into the origin of three

  18. Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

    Energy Technology Data Exchange (ETDEWEB)

    Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

    1988-09-26

    The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

  19. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    Science.gov (United States)

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  20. Copper intoxication inhibits aerobic nucleotide synthesis in Streptococcus pneumoniae

    Science.gov (United States)

    Johnson, Michael D. L.; Kehl-Fie, Thomas E.; Rosch, Jason W.

    2015-01-01

    Copper is universally toxic in excess, a feature exploited by the human immune system to facilitate bacterial clearance. The mechanism of copper intoxication remains unknown for many bacterial species. Here, we demonstrate that copper toxicity in Streptococcus pneumoniae is independent from oxidative stress but, rather, is the result of copper inhibiting the aerobic dNTP biosynthetic pathway. Furthermore, we show that copper-intoxicated S. pneumoniae is rescued by manganese, which is an essential metal in the aerobic nucleotide synthesis pathway. These data provide insight into new targets to enhance copper-mediated toxicity during bacterial clearance. PMID:25730343

  1. Genome-wide patterns of nucleotide polymorphism in domesticated rice

    DEFF Research Database (Denmark)

    Caicedo, Ana L; Williamson, Scott H; Hernandez, Ryan D;

    2007-01-01

    to explain contemporary patterns of polymorphisms in rice, including a (i) selectively neutral population bottleneck model, (ii) bottleneck plus migration model, (iii) multiple selective sweeps model, and (iv) bottleneck plus selective sweeps model. We find that a simple bottleneck model, which has been...... the dominant demographic model for domesticated species, cannot explain the derived nucleotide polymorphism site frequency spectrum in rice. Instead, a bottleneck model that incorporates selective sweeps, or a more complex demographic model that includes subdivision and gene flow, are more plausible...

  2. Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures.

    Science.gov (United States)

    Abi, Alireza; Ferapontova, Elena E

    2013-04-01

    Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.

  3. ATCG nucleotide fluctuation of Deinococcus radiodurans radiation genes

    Science.gov (United States)

    Holden, Todd; Subramaniam, R.; Sullivan, R.; Cheung, E.; Schneider, C.; Tremberger, G., Jr.; Flamholz, A.; Lieberman, D. H.; Cheung, T. D.

    2007-09-01

    The radiation resistance-repair genes in Deinococcus radiodurans (DR) and E-coli were analyzed in terms of the A, T, C, G nucleotide fluctuations. The studied genes were Rec-A, Rec-Q, and the unique DR PprA gene. In an ATCG sequence, each base was assigned a number equal to its atomic number. The resulting numerical sequence was the basis of the statistical analysis. Fractal analysis using the Higuchi method gave a fractal dimension increase of the Deinococcus radiodurans genes as compared to E-coli, which is comparable to the enhancement observed in the human HAR1 region (HAR1F gene) over that of the chimpanzee. Near neighbor fluctuation was also studied via the Black-Scholes model where the increment sequence was treated as a random walk series. The Deinococcus radiodurans radiation gene standard deviations were consistently higher than that of the E-coli deviations, and agree with the fractal analysis results. The sequence stacking interaction was studied using the published nucleotide-pair melting free energy values and Deinococcus radiodurans radiation genes were shown to possess larger negative free energies. The high sensitivity of the fractal dimension as a biomarker was tested with correlation analysis of the gamma ray dose versus fractal dimension, and the R square values were found to be above 0.9 (N=5). When compared with other nucleotide sequences such as the rRNA sequences, HAR1 and its chimpanzee counterpart, the higher fluctuation (correlated randomness) and larger negative free energy of a DR radiation gene suggested that a radiation resistance-repair sequence exhibited higher complexity. As the HAR1 nucleotide sequence complexity and its transcription activity of co-expressing cortex protein reelin supported a positive selection event in humans, a similar inference of positive selection of coding genes could be drawn for Deinococcus radiodurans when compared to E-coli. The origin of such a positive selection would be consistent with that of a

  4. Old concepts, new molecules and current approaches applied to the bacterial nucleotide signalling field.

    Science.gov (United States)

    Gründling, Angelika; Lee, Vincent T

    2016-11-01

    Signalling nucleotides are key molecules that help bacteria to rapidly coordinate cellular pathways and adapt to changes in their environment. During the past 10 years, the nucleotide signalling field has seen much excitement, as several new signalling nucleotides have been discovered in both eukaryotic and bacterial cells. The fields have since advanced quickly, aided by the development of important tools such as the synthesis of modified nucleotides, which, combined with sensitive mass spectrometry methods, allowed for the rapid identification of specific receptor proteins along with other novel genome-wide screening methods. In this review, we describe the principle concepts of nucleotide signalling networks and summarize the recent work that led to the discovery of the novel signalling nucleotides. We also highlight current approaches applied to the research in the field as well as resources and methodological advances aiding in a rapid identification of nucleotide-specific receptor proteins.This article is part of the themed issue 'The new bacteriology'. PMID:27672152

  5. Electrochemically-tuned luminescence of a [Ru(bpy) 2(tatp)] 2+-sensitized TiO 2 anode and its applications to photo-stimulated guanine/H 2O 2 fuel cells

    Science.gov (United States)

    Shao, Jiangyang; Sun, Ting; Ji, Shibo; Li, Hong; Lan, Sheng; Li, Weishan

    A phenazine-containing Ru(II) complex [Ru(bpy) 2(tatp)] 2+ (bpy = 2,2‧-bipyridine and tatp = 1,4,8,9-tetra-aza-triphenylene) is first applied to a modification of the nano-TiO 2/indium-tin oxide (ITO) electrode by the method of repetitive voltammetric sweeping. The resulting [Ru(bpy) 2(tatp)] 2+-modified TiO 2 electrode shows two pairs of well-defined redox waves and excellent electrocatalytic activity for the oxidation of guanine. [Ru(bpy) 2(tatp)] 2+ on TiO 2 surfaces exhibits intense absorbance and photoluminescence in visible region, revealed by absorption spectra, emission spectra and fluorescence microscope. While [Ru(bpy) 2(tatp)] 2+-sensitized TiO 2 is functionalized as an anode to combine with a continuous wave green laser via an optical microscope, the luminescence of Ru(II)-based excited states can be enhanced by the oxidation of guanine. Furthermore, the [Ru(bpy) 2(tatp)] 2+-sensitized TiO 2 electrode is used as photoanode and hemoglobin-modified single-walled carbon nanotubes (SWCNTs) as cathode for the elaboration of a photo-stimulated guanine/H 2O 2 fuel cell with a saturated KCl salt-bridge. It becomes evident that the photo-stimulated fuel cell performance depends strongly on the excited states of Ru(II) complex-sensitized anodes as well as the electrocatalytic oxidation of guanine. This study provides an electrochemically-tuned luminescence method for better evaluating contributions of the sensitizer excited states to photo-stimulated fuel cells.

  6. MS-CASPT2 study of hole transfer in guanine-indole complexes using the generalized Mulliken-Hush method: effective two-state treatment.

    Science.gov (United States)

    Butchosa, C; Simon, S; Blancafort, L; Voityuk, A

    2012-07-12

    Because hole transfer from nucleobases to amino acid residues in DNA-protein complexes can prevent oxidative damage of DNA in living cells, computational modeling of the process is of high interest. We performed MS-CASPT2 calculations of several model structures of π-stacked guanine and indole and derived electron-transfer (ET) parameters for these systems using the generalized Mulliken-Hush (GMH) method. We show that the two-state model commonly applied to treat thermal ET between adjacent donor and acceptor is of limited use for the considered systems because of the small gap between the ground and first excited states in the indole radical cation. The ET parameters obtained within the two-state GMH scheme can deviate significantly from the corresponding matrix elements of the two-state effective Hamiltonian based on the GMH treatment of three adiabatic states. The computed values of diabatic energies and electronic couplings provide benchmarks to assess the performance of less sophisticated computational methods.

  7. Activation of guanine-{beta}-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

    Energy Technology Data Exchange (ETDEWEB)

    Leanza, Luigi; Miazzi, Cristina; Ferraro, Paola; Reichard, Peter; Bianchi, Vera, E-mail: vbianchi@bio.unipd.it

    2010-12-10

    The deoxyguanosine (GdR) analog guanine-ss-D-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

  8. 2-Alkynyl-N-propargyl pyridinium salts: pyridinium-based heterocyclic skipped aza-enediynes that cleave DNA by deoxyribosyl hydrogen-atom abstraction and guanine oxidation.

    Science.gov (United States)

    Tuesuwan, Bodin; Kerwin, Sean M

    2006-06-13

    Diradical-generating cyclizations such as the enediyne Bergman cyclization and the enyne allene Myers-Saito cyclization have been exploited by nature in the mechanism of DNA cleavage by a series of potent antitumor antibiotics. Alternative diradical-generating cyclizations have been proposed in the design of selective antitumor agents; however, little information is available concerning the utility of these alternative cyclizations in radical-based DNA cleavage chemistry. One such alternative diradical-generating cyclization, the aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes, has been recently reported. Here, we report the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-N-propargyl pyridinium salts). Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. Within this series of compounds, the analogue bearing a p-methoxyphenyl group on the pyridinium 2-alkyne substituents was found to be the most effective DNA cleavage agent, displaying significant supercoiled DNA-nicking activity at concentrations as low as 1 microM. Detailed studies of this analogue show that DNA cleavage occurs through 4'-hydrogen-atom abstraction from the DNA backbone and oxidation of guanine bases. This is the first report of enediyne-like radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization. PMID:16752915

  9. Determination of guanine and adenine by high-performance liquid chromatography with a self-fabricated wall-jet/thin-layer electrochemical detector at a glassy carbon electrode.

    Science.gov (United States)

    Zhou, Yaping; Yan, Hongling; Xie, Qingji; Yao, Shouzhuo

    2015-03-01

    A sensitive wall-jet/thin-layer amperometric electrochemical detector (ECD) coupled to high-performance liquid chromatography (HPLC) was developed for simultaneous determination of guanine (G) and adenine (A). The analytes were detected at a glassy carbon electrode (GCE) and the HPLC-ECD calibration curves showed good linearity (R(2)>0.997) under optimized conditions. Limits of detection for G and A are 0.6 nM and 1.4 nM (S/N=3), respectively, which are lower than those obtained with an UV-vis detector and a commercial electrochemical detector. We have successfully applied this HPLC-ECD to assess the contents of G and A in hydrochloric acid-digested calf thymus double-stranded DNA. In addition, we compared in detail the analysis of G and A by cyclic voltammetry (CV) and by the HPLC-ECD system on both bare GCE and electroreduced graphene oxide (ERGO) modified GCE. We found that the adsorption of G and A on the electrode surfaces can vary their anodic CV peaks and the competitive adsorption of G and A on the limited sites of the electrode surfaces can cause crosstalk effects on their anodic CV peak signals, but the HPLC-ECD system is insensitive to such electrode-adsorption and can give more reliable analytical results. PMID:25618679

  10. Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    Science.gov (United States)

    Terato, Hiroaki; Masaoka, Aya; Asagoshi, Kenjiro; Honsho, Akiko; Ohyama, Yoshihiko; Suzuki, Toshinori; Yamada, Masaki; Makino, Keisuke; Yamamoto, Kazuo; Ide, Hiroshi

    2002-01-01

    Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (Vmax/Km) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [3H]Xan from the substrate. The treatment of E.coli with N-methyl-N′-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA+ but not alkA– strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan. PMID:12434002

  11. Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

    Science.gov (United States)

    Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

    2014-09-01

    Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite. PMID:24923662

  12. Detecting Single-Nucleotide Substitutions Induced by Genome Editing.

    Science.gov (United States)

    Miyaoka, Yuichiro; Chan, Amanda H; Conklin, Bruce R

    2016-01-01

    The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells.

  13. Single nucleotide polymorphism analysis of European archaeological M. leprae DNA.

    Directory of Open Access Journals (Sweden)

    Claire L Watson

    Full Text Available BACKGROUND: Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA from medieval bones and single nucleotide polymorphism (SNP typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution. METHODS AND FINDINGS: Skeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia and are dated around the medieval period (476 to 1350 A.D.. we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3. CONCLUSIONS: These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.

  14. Single Nucleotide Polymorphism Analysis of Protamine Genes in Infertile Men

    Directory of Open Access Journals (Sweden)

    Ahamad Salamian

    2008-01-01

    Full Text Available Background: Single nucleotide polymorphism (SNPs are considered as one of the underlyingcauses of male infertility. Proper sperm chromatin packaging which involves replacement ofhistones with protamines has profound effect on male fertility. Over 20 SNPs have been reportedfor the protamine 1 and 2.Materials and Methods: The aim of this study was to evaluate the frequency of two previouslyreported SNPs using polymerase chain reaction (PCR-restriction fragment length polymorphism(RFLP approach in 35, 96 and 177 normal, oligozoospermic and azoospermic individuals. TheseSNPs are: 1. A base pair substitution (G at position 197 instead of T in protamine type 1 Openreading frame (ORF including untranslated region, which causes an Arg residue change to Serresidue in a highly conserved region. 2. cytidine nucleotide change to thymidine in position of 248of protamine type 2 ORF which caused a nonsense point mutation.Results: The two mentioned SNPs were not present in the studied population, thus concluding thatthese SNPs can not serves as molecular markers for male infertility diagnosis.Conclusion: The results of our study reveal that in a selected Iranian population, the SNP G197Tand C248T are completely absent and are not associated with male infertility and therefore theseSNPs may not represent a molecular marker for genetic diagnosis of male infertility.

  15. Allosteric interactions of DNA and nucleotides with S. cerevisiae RSC.

    Science.gov (United States)

    Malik, Shuja Shafi; Rich, Evan; Viswanathan, Ramya; Cairns, Bradley R; Fischer, Christopher J

    2011-09-20

    RSC (remodel the structure of chromatin) is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a "trimeric minimal RSC" (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein-labeled double-stranded DNA with a K(d) of ∼100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration-dependent manner with the addition of AMP-PNP having more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affects the affinity of DNA binding by RSCt suggest that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the apparent low processivity of DNA translocation by the enzyme.

  16. Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

    Science.gov (United States)

    Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun

    2015-11-10

    Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.

  17. Enzymatic polymerisation involving 2'-amino-LNA nucleotides.

    Science.gov (United States)

    Johannsen, Marie W; Veedu, Rakesh N; Madsen, Andreas Stahl; Wengel, Jesper

    2012-05-15

    The triphosphate of the thymine derivative of 2'-amino-LNA (2'-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2'-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2'-Amino-LNA-T in the template and 2'-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2'-amino-LNA-T nucleotides. PMID:22503454

  18. Computational learning on specificity-determining residue-nucleotide interactions

    KAUST Repository

    Wong, Ka-Chun

    2015-11-02

    The protein–DNA interactions between transcription factors and transcription factor binding sites are essential activities in gene regulation. To decipher the binding codes, it is a long-standing challenge to understand the binding mechanism across different transcription factor DNA binding families. Past computational learning studies usually focus on learning and predicting the DNA binding residues on protein side. Taking into account both sides (protein and DNA), we propose and describe a computational study for learning the specificity-determining residue-nucleotide interactions of different known DNA-binding domain families. The proposed learning models are compared to state-of-the-art models comprehensively, demonstrating its competitive learning performance. In addition, we describe and propose two applications which demonstrate how the learnt models can provide meaningful insights into protein–DNA interactions across different DNA binding families.

  19. Nucleotide sequence of Streptomyces griseus initiator tRNA.

    OpenAIRE

    Kuchino, Y; Yamamoto, I.; Nishimura, S.

    1982-01-01

    The primary structure of initiator tRNA from Streptomyces griseus was determined by post-labeling procedures. The nucleotide sequence is pC-G-C-G-G-G-G-U-G-G-A-G-C-A-G-C-U-C-G-G-D-A-G-C-U-C-G-C-U-G-G-G-C-U-C-A-U-A-A-C-C- C-A-G-A-G-G-U-C-G-C-A-G-G-U-psi-C-A-m1A-A-U-C-C-U-G-U-C-C-C-C-G-C-U-A-C-C-A0H. The unique feature of the sequence of this tRNA is that residue 54 is occupied by unmodified U, while ribothymidine is located in that position in most initiator tRNAs from eubacteria.

  20. Countermeasure for space flight effects on immune system: nutritional nucleotides

    Science.gov (United States)

    Kulkarni, A. D.; Yamauchi, K.; Sundaresan, A.; Ramesh, G. T.; Pellis, N. R.

    2005-01-01

    Microgravity and its environment have adverse effects on the immune system. Abnormal immune responses observed in microgravity may pose serious consequences, especially for the recent directions of NASA for long-term space missions to Moon, Mars and deep Space exploration. The study of space flight immunology is limited due to relative inaccessibility, difficulty of performing experiments in space, and inadequate provisions in this area in the United States and Russian space programs (Taylor 1993). Microgravity and stress experienced during space flights results in immune system aberration (Taylor 1993). In ground-based mouse models for some of the microgravity effects on the human body, hindlimb unloading (HU) has been reported to cause abnormal cell proliferation and cytokine production (Armstrong et al., 1993, Chapes et al. 1993). In this report, we document that a nutritional nucleotide supplementation as studied in ground-based microgravity analogs, has potential to serve as a countermeasure for the immune dysfunction observed in space travel.

  1. Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and RNA.

    Science.gov (United States)

    Samanta, Biswajit; Seikowski, Jan; Höbartner, Claudia

    2016-01-26

    5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA. PMID:26679556

  2. A nucleotide-level coarse-grained model of RNA

    CERN Document Server

    Šulc, Petr; Ouldridge, Thomas E; Doye, Jonathan P K; Louis, Ard A

    2014-01-01

    We present a new, nucleotide-level model for RNA, oxRNA, based on the coarse-graining methodology recently developed for the oxDNA model of DNA. The model is designed to reproduce structural, mechanical and thermodynamic properties of RNA, and the coarse-graining level aims to retain the relevant physics for RNA hybridization and the structure of single- and double-stranded RNA. In order to explore its strengths and weaknesses, we test the model in a range of nanotechnological and biological settings. Applications explored include the folding thermodynamics of a pseudoknot, the formation of a kissing loop complex, the structure of a hexagonal RNA nanoring, and the unzipping of a hairpin motif. We argue that the model can be used for efficient simulations of the structure of systems with thousands of base pairs, and for the assembly of systems of up to hundreds of base pairs. The source code implementing the model is released for public use.

  3. Computational identification of candidate nucleotide cyclases in higher plants

    KAUST Repository

    Wong, Aloysius Tze

    2013-09-03

    In higher plants guanylyl cyclases (GCs) and adenylyl cyclases (ACs) cannot be identified using BLAST homology searches based on annotated cyclic nucleotide cyclases (CNCs) of prokaryotes, lower eukaryotes, or animals. The reason is that CNCs are often part of complex multifunctional proteins with different domain organizations and biological functions that are not conserved in higher plants. For this reason, we have developed CNC search strategies based on functionally conserved amino acids in the catalytic center of annotated and/or experimentally confirmed CNCs. Here we detail this method which has led to the identification of >25 novel candidate CNCs in Arabidopsis thaliana, several of which have been experimentally confirmed in vitro and in vivo. We foresee that the application of this method can be used to identify many more members of the growing family of CNCs in higher plants. © Springer Science+Business Media New York 2013.

  4. The nucleotide sequence of the bacteriophage T5 ltf gene.

    Science.gov (United States)

    Kaliman, A V; Kulshin, V E; Shlyapnikov, M G; Ksenzenko, V N; Kryukov, V M

    1995-06-01

    The nucleotide sequence of the bacteriophage T5 Bg/II-BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L-shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine-Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage lambda in a segment of 118 amino acids overlap. PMID:7789514

  5. Milestones in the discovery of antiviral agents: nucleosides and nucleotides

    Directory of Open Access Journals (Sweden)

    Erik de Clercq

    2012-12-01

    Full Text Available In this review article, a number of milestones in the antiviral research field on nucleosides and nucleotides are reviewed in which the author played a significant part, especially in the initial stages of their development. Highlighted are the amino acyl esters of acyclovir, particularly valacyclovir (VACV, brivudin (BVDU and the valine ester of Cf1743 (FV-100, the 2′,3′-dideoxynucleosides (nucleoside reverse transcriptase inhibitors, NRTIs, the acyclic nucleoside phosphonates (S-HPMPA, (S-HPMPC (cidofovir and alkoxyalkyl esters thereof (HDP-, ODE-CDV, adefovir and adefovir dipivoxil, tenofovir and tenofovir disoproxil fumarate (TDF, combinations containing TDF and emtricitabine, i.e., Truvada®, Atripla®, Complera®/Eviplera® and the Quad pill, and the phosphonoamidate derivatives GS-7340, GS-9131, GS-9191 and GS-9219.

  6. Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guintini, Laetitia; Charton, Romain; Peyresaubes, François; Thoma, Fritz; Conconi, Antonio

    2015-12-01

    The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.

  7. Current research status, databases and application of single nucleotide polymorphism.

    Science.gov (United States)

    Javed, R; Mukesh

    2010-07-01

    Single Nucleotide Polymorphisms (SNPs) are the most frequent form of DNA variation in the genome. SNPs are genetic markers which are bi-allelic in nature and grow at a very fast rate. Current genomic databases contain information on several million SNPs. More than 6 million SNPs have been identified and the information is publicly available through the efforts of the SNP Consortium and others data bases. The NCBI plays a major role in facillating the identification and cataloging of SNPs through creation and maintenance of the public SNP database (dbSNP) by the biomedical community worldwide and stimulate many areas of biological research including the identification of the genetic components of disease. In this review article, we are compiling the existing SNP databases, research status and their application. PMID:21717869

  8. Efficient fidelity control by stepwise nucleotide selection in polymerase elongation

    CERN Document Server

    Yu, Jin

    2014-01-01

    Polymerases select nucleotides before incorporating them for chemical synthesis during gene replication or transcription. How the selection proceeds stepwise efficiently to achieve sufficiently high fidelity and speed is essential for polymerase function. We examined step-by-step selections that have conformational transition rates tuned one at time in the polymerase elongation cycle, with a controlled differentiation free energy at each checkpoint. The elongation is sustained at non-equilibrium steady state with constant free energy input and heat dissipation. It is found that error reduction capability does not improve for selection checkpoints down the reaction path. Hence, it is essential to select early to achieve an efficient fidelity control. In particular, for two consecutive selections that reject the wrong substrate back and inhibit it forward from a same kinetic state, the same error rates are obtained at the same free energy differentiation. The initial screening is indispensible for maintaining t...

  9. Gene : CBRC-CJAC-01-1151 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available : Rho guanine nucleotide exchange factor 10 [Pan troglodytes] 7e-08 25% MGIGWVLVHVSFMGWVLDGYWCVFHGMGTGWVLVCV...SWDGYWMGTGVCFIHGMGSGWVLVCVSFMGWVLDGYWCVFHSWDGYWLGTGACVIHGMGTGWVLVCVSFMGWVPDGYWCVFHSWDGFWMGTGVCFIHGMGTGWVLVCVSFM...VWVLDGYWCVCHSWDGYWCVLSWDGNWMGTSVCFIHGMGTGAYVIHGMGAGWVLVHVSFMVWVLDGYWYVFHSWDGHWMGTGVCFIHGMGTGWVLVCVSFM...GWVLDGYWCVFHGMGTGWVLVCVSFMGWALDGYWCVCHSWDGHWMGTGVCVIHGMGTGWVLVCVSFMGWVLDGYLCVCHSWDGYWMGTGVCVIPGMGTCV ...

  10. A model for the evolution of nucleotide polymerase directionality.

    Directory of Open Access Journals (Sweden)

    Joshua Ballanco

    Full Text Available BACKGROUND: In all known living organisms, every enzyme that synthesizes nucleic acid polymers does so by adding nucleotide 5′-triphosphates to the 3′-hydroxyl group of the growing chain. This results in the well known 5'→3' directionality of all DNA and RNA Polymerases. The lack of any alternative mechanism, e.g. addition in a 3'→5' direction, may indicate a very early founder effect in the evolution of life, or it may be the result of a selective pressure against such an alternative. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to determine whether the lack of an alternative polymerase directionality is the result of a founder effect or evolutionary selection, we have constructed a basic model of early polymerase evolution. This model is informed by the essential chemical properties of the nucleotide polymerization reaction. With this model, we are able to simulate the growth of organisms with polymerases that synthesize either 5'→3' or 3'→5' in isolation or in competition with each other. CONCLUSIONS/SIGNIFICANCE: We have found that a competition between organisms with 5'→3' polymerases and 3'→5' polymerases only results in a evolutionarily stable strategy under certain conditions. Furthermore, we have found that mutations lead to a much clearer delineation between conditions that lead to a stable coexistence of these populations and conditions which ultimately lead to success for the 5'→3' form. In addition to presenting a plausible explanation for the uniqueness of enzymatic polymerization reactions, we hope these results also provide an example of how whole organism evolution can be understood based on molecular details.

  11. Morphine enhances purine nucleotide catabolism in rive and in vitro

    Institute of Scientific and Technical Information of China (English)

    Chang LIU; Jian-kai LIU; Mu-jie KAN; Lin GAO; Hai-ying FU; Hang ZHOU; Min HONG

    2007-01-01

    Aim: To investigate the effect and mechanism of morphine on purine nucleotide catabolism. Methods: The rat model of morphine dependence and withdrawal and rat C6 glioma cells in culture were used. Concentrations of uric acid in the plasma were measured by the uricase-rap method, adenosine deaminase (ADA) and xan- thine oxidase (XO) in the plasma and tissues were measured by the ADA and XO test kit. RT-PCR and RT-PCR-Southern blotting were used to examine the relative amount of ADA and XO gene transcripts in tissues and C6 cells. Results: (i) the concentration of plasma uric acid in the morphine-administered group was signifi-cantly higher (P<0.05) than the control group; (ii) during morphine administration and withdrawal periods, the ADA and XO concentrations in the plasma increased significantly (P<0.05); (iii) the amount of ADA and XO in the parietal lobe, liver, small intestine, and skeletal muscles of the morphine-administered groups increased, while the level of ADA and XO in those tissues of the withdrawal groups decreased; (iv) the transcripts of the ADA and XO genes in the parietal lobe, liver, small intestine, and skeletal muscles were higher in the morphine-administered group. The expression of the ADA and XO genes in those tissues returned to the control level during morphine withdrawal, with the exception of the skeletal muscles; and (v) the upregulation of the expression of the ADA and XO genes induced by morphine treatment could be reversed by naloxone. Conclusion: The effects of morphine on purine nucleotide metabolism might be an important, new biochemical pharmacological mechanism of morphine action.

  12. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    Science.gov (United States)

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  13. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  14. Prodrugs of the selective antiherpesvirus agent 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (BRL 39123) with improved gastrointestinal absorption properties.

    Science.gov (United States)

    Harnden, M R; Jarvest, R L; Boyd, M R; Sutton, D; Vere Hodge, R A

    1989-08-01

    Potential oral prodrugs of the antiherpesvirus acyclonucleoside 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (1, BRL 39123) have been synthesized and evaluated for bioavailability of 1 in the blood of mice. Reduction of 9-[4-acetoxy-3-(acetoxymethyl)but-1-yl]-2-amino-6-chloropurine (13) using ammonium formate and 10% palladium on carbon afforded the 2-aminopurine 14, which was hydrolyzed to the monoacetate 15 and to 2-amino-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purine (5). The 2-aminopurine 5 was subsequently converted to additional monoester (17, 21-23) and diester (16, 24) derivatives and to its di-O-isopropylidene derivative 18. Both 5 and its esters (14-17, 21, 22) and also 18 were well absorbed after oral administration and converted efficiently to 1, the diacetyl (14) and dipropionyl (16) esters providing concentrations of 1 in the blood that were more than 15-fold higher than those observed after dosing either 1 or its esters (25-27). Some 6-alkoxy-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purines (8-10), the preparation of which has been reported previously, also showed improved absorption properties, but their conversion to 1 was less efficient than for the 2-aminopurine derivatives. On the basis of these results and subsequent experiments involving determinations of rates of conversion to 1 in the presence of rat and human tissue preparations, 9-[4-acetoxy-3-(acetoxymethyl)but-1-yl]-2-aminopurine (14, BRL 42810) was identified as the preferred prodrug of 1. Oral bioavailability studies in healthy human subjects confirmed 14 as an effective prodrug, and this compound is now being evaluated in clinical trials. PMID:2754699

  15. Electronic absorption, vibrational spectra, non-linear optical properties, NBO analysis and thermodynamic properties of 9-[(2-hydroxyethoxy) methyl] guanine molecule by density functional method

    Science.gov (United States)

    Rajamani, T.; Muthu, S.

    2013-02-01

    The Fourier transform infrared (FT-IR) and FT-Raman of 9-[(2-hydroxyethoxy) methyl] guanine (9-2HEMG) have been recorded in the regions 4000-100 and 4000-400 cm-1, respectively. A complete assignment and analysis of the fundamental vibrational modes of the molecule were carried out. The observed fundamental modes have been compared with the harmonic vibrational frequencies computed using DFT (B3LYP) method by employing 6-31G(d,p) and 3-21G basis sets. The vibrational studies were interpreted in terms of potential energy distribution. The first order hyperpolarizability (β0) and related properties (α, μ and Δα) of this molecular system are calculated using B3LYP/6-31G(d,p) method based on the finite-field approach. Stability of the molecule arising from hyperconjugative interactions and charge delocalization has been analyzed using natural bond orbital (NBO) analysis. The results show that electron density (ED) in the σ* and π* antibonding orbitals and second-order delocalization energies (E(2)) confirm the occurrence of intramolecular charge transfer (ICT) within the molecule. UV-vis spectrum of the compound has been recorded and electronic properties such as excitation energies, oscillator strength and wavelength are calculated by TD-DFT and CIS methods using B3LYP/6-31G (d,p) basis set. Molecular electrostatic potential (MEP) and HOMO-LUMO energy levels are also constructed. The thermodynamic properties of the title compound have been calculated at different temperatures and the results reveal that the standard heat capacities (Cp,m), standard entropies (Sm) and standard enthalpy changes (Hm) increase with rise in temperature.

  16. The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus.

    OpenAIRE

    1984-01-01

    The complete nucleotide sequence of the coding region of foot and mouth disease virus RNA (strain A1061) is presented. The sequence extends from the primary initiation site, approximately 1200 nucleotide from the 5' end of the genome, in an open translational reading frame of 6,999 nucleotides to a termination codon 93 nucleotides from the 3' terminal poly (A). Available amino acid sequence data correlates with that predicted from the nucleotide sequence. The amino acid sequence around cleava...

  17. 77 FR 65537 - Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence...

    Science.gov (United States)

    2012-10-29

    ... Amino Acid Sequence Disclosures ACTION: Proposed collection; comment request. SUMMARY: The United States....'' SUPPLEMENTARY INFORMATION: I. Abstract Patent applications that contain nucleotide and/or amino acid...

  18. Enzymatic synthesis of radioisotope-labeled nucleotides from the corresponding labeled nitrogen bases

    International Nuclear Information System (INIS)

    The authors have developed a procedure that permits the production of nucleotides multiply labeled with various radioisotopes, with a molar radioactivity equal to the molar radioactivity of the original nitrogen bases. The methods of isolation and purification of the enzyme preparations were studied on the enzyme systems converting nitrogen bases to nucleoside triphosphates in the presence of phosphoribosyl pyrophosphate or ribose-5-phosphate. The presence of nucleotide impurities in the enzyme preparation was determined spectrophotometrically. The authors were able to practically avoid nucleotide impurities by salting out the protein fraction of interest with ammonium sulfate. The authors succeeded in reducing the content of nucleotides in the enzyme preparation by a factor of 40

  19. DNA Methylation is Associated with an Increased Level of Conservation at Nondegenerate Nucleotides in Mammals

    Science.gov (United States)

    Chuang, Trees-Juen; Chen, Feng-Chi

    2014-01-01

    DNA methylation at CpG dinucleotides can significantly increase the rate of cytosine-to-thymine mutations and the level of sequence divergence. Although the correlations between DNA methylation and genomic sequence evolution have been widely studied, an unaddressed yet fundamental question is how DNA methylation is associated with the conservation of individual nucleotides in different sequence contexts. Here, we demonstrate that in mammalian exons, the correlations between DNA methylation and the conservation of individual nucleotides are dependent on the type of exonic sequence (coding or untranslated), the degeneracy of coding nucleotides, background selection pressure, and the relative position (first or nonfirst exon in the transcript) where the nucleotides are located. For untranslated and nonzero-fold degenerate nucleotides, methylated sites are less conserved than unmethylated sites regardless of background selection pressure and the relative position of the exon. For zero-fold degenerate (or nondegenerate) nucleotides, however, the reverse trend is observed in nonfirst coding exons and first coding exons that are under stringent background selection pressure. Furthermore, cytosine-to-thymine mutations at methylated zero-fold degenerate nucleotides are predicted to be more detrimental than those that occur at unmethylated nucleotides. As zero-fold and nonzero-fold degenerate nucleotides are very close to each other, our results suggest that the “functional resolution” of DNA methylation may be finer than previously recognized. In addition, the positive correlation between CpG methylation and the level of conservation at zero-fold degenerate nucleotides implies that CpG methylation may serve as an “indicator” of functional importance of these nucleotides. PMID:24157417

  20. Energy efficiency trade-offs drive nucleotide usage in transcribed regions

    Science.gov (United States)

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J.

    2016-01-01

    Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of ‘A' versus ‘T' and ‘G' versus ‘C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides ‘U' and ‘C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides ‘A' and ‘G' at non-synonymous coding sites. PMID:27098217

  1. Energy efficiency trade-offs drive nucleotide usage in transcribed regions.

    Science.gov (United States)

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J

    2016-04-21

    Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of 'A' versus 'T' and 'G' versus 'C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides 'U' and 'C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides 'A' and 'G' at non-synonymous coding sites.

  2. Single Nucleotide Polymorphism Clustering in Systemic Autoimmune Diseases

    Science.gov (United States)

    Charlon, Thomas; Bossini-Castillo, Lara; Carmona, F. David; Di Cara, Alessandro; Wojcik, Jérôme; Voloshynovskiy, Sviatoslav

    2016-01-01

    Systemic Autoimmune Diseases, a group of chronic inflammatory conditions, have variable symptoms and difficult diagnosis. In order to reclassify them based on genetic markers rather than clinical criteria, we performed clustering of Single Nucleotide Polymorphisms. However naive approaches tend to group patients primarily by their geographic origin. To reduce this “ancestry signal”, we developed SNPClust, a method to select large sources of ancestry-independent genetic variations from all variations detected by Principal Component Analysis. Applied to a Systemic Lupus Erythematosus case control dataset, SNPClust successfully reduced the ancestry signal. Results were compared with association studies between the cases and controls without or with reference population stratification correction methods. SNPClust amplified the disease discriminating signal and the ratio of significant associations outside the HLA locus was greater compared to population stratification correction methods. SNPClust will enable the use of ancestry-independent genetic information in the reclassification of Systemic Autoimmune Diseases. SNPClust is available as an R package and demonstrated on the public Human Genome Diversity Project dataset at https://github.com/ThomasChln/snpclust. PMID:27490238

  3. Nucleotide sequence determines the accelerated rate of point mutations.

    Science.gov (United States)

    Kini, R Manjunatha; Chinnasamy, Arunkumar

    2010-09-01

    Although the theory of evolution was put forth about 150 years ago our understanding of how molecules drive evolution remains poor. It is well-established that proteins evolve at different rates, essentially based on their functional role and three-dimensional structure. However, the highly variable rates of evolution of different proteins - especially the rapidly evolving ones - within a single organism are poorly understood. Using examples of genes for fast-evolving toxins and human hereditary diseases, we show for the first time that specific nucleotide sequences appear to determine point mutation rates. Based on mutation rates, we have classified triplets (not just codons) into stable, unstable and intermediate groups. Toxin genes contain a relatively higher percentage of unstable triplets in their exons compared to introns, whereas non-toxin genes contain a higher percentage of unstable triplets in their introns. Thus the distribution of stable and unstable triplets is correlated with and may explain the accelerated evolution of point mutations in toxins. Similarly, at the genomic level, lower organisms with genes that evolve faster contain a higher percentage of unstable triplets compared to higher organisms. These findings show that mutation rates of proteins, and hence of the organisms, are DNA sequence-dependent and thus provide a proximate mechanism of evolution at the molecular level. PMID:20362603

  4. ADH single nucleotide polymorphism associations with alcohol metabolism in vivo

    Science.gov (United States)

    Birley, Andrew J.; James, Michael R.; Dickson, Peter A.; Montgomery, Grant W.; Heath, Andrew C.; Martin, Nicholas G.; Whitfield, John B.

    2009-01-01

    We have previously found that variation in alcohol metabolism in Europeans is linked to the chromosome 4q region containing the ADH gene family. We have now typed 103 single nucleotide polymorphisms (SNPs) across this region to test for allelic associations with variation in blood and breath alcohol concentrations after an alcohol challenge. In vivo alcohol metabolism was modelled with three parameters that identified the absorption and rise of alcohol concentration following ingestion, and the rate of elimination. Alleles of ADH7 SNPs were associated with the early stages of alcohol metabolism, with additional effects in the ADH1A, ADH1B and ADH4 regions. Rate of elimination was associated with SNPs in the intragenic region between ADH7 and ADH1C, and across ADH1C and ADH1B. SNPs affecting alcohol metabolism did not correspond to those reported to affect alcohol dependence or alcohol-related disease. The combined SNP associations with early- and late-stage metabolism only account for approximately 20% of the total genetic variance linked to the ADH region, and most of the variance for in vivo alcohol metabolism linked to this region is yet to be explained. PMID:19193628

  5. Unique nucleotide polymorphism of ankyrin gene cluster in Arabidopsis

    Indian Academy of Sciences (India)

    Jianchang Du; Xingna Wang; Mingsheng Zhang; Dacheng Tian; Yong-Hua Yang

    2007-01-01

    The ankyrin (ANK) gene cluster is a part of a multigene family encoding ANK transmembrane proteins in Arabidopsis thaliana, and plays an important role in protein–protein interactions and in signal pathways. In contrast to other regions of a genome, the ANK gene cluster exhibits an extremely high level of DNA polymorphism in an ∼5-kb region, without apparent decay. Phylogenetic analysis detects two clear, deeply differentiated haplotypes (dimorphism). The divergence between haplotypes of accession Col-0 and Ler-0 (Hap-C and Hap-L) is estimated to be 10.7%, approximately equal to the 10.5% average divergence between A. thaliana and A. lyrata. Sequence comparisons for the ANK gene cluster homologues in Col-0 indicate that the members evolve independently, and that the similarity among paralogues is lower than between alleles. Very little intralocus recombination or gene conversion is detected in ANK regions. All these characteristics of the ANK gene cluster are consistent with a tandem gene duplication and birth-and-death process. The possible mechanisms for and implications of this elevated nucleotide variation are also discussed, including the suggestion of balancing selection.

  6. Bulk segregant analysis using single nucleotide polymorphism microarrays.

    Directory of Open Access Journals (Sweden)

    Anthony Becker

    Full Text Available Bulk segregant analysis (BSA using microarrays, and extreme array mapping (XAM have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species. These experiments, however, require the identification of single feature polymorphisms (SFP between the cross parents for each new combination of genotypes, which raises the cost of experiments. The availability of the genomic polymorphism data in Arabidopsis thaliana, coupled with the efficient designs of Single Nucleotide Polymorphism (SNP genotyping arrays removes the requirement for SFP detection and lowers the per array cost, thereby lowering the overall cost per experiment. To demonstrate that these approaches would be functional on SNP arrays and determine confidence intervals, we analyzed hybridizations of natural accessions to the Arabidopsis ATSNPTILE array and simulated BSA or XAM given a variety of gene models, populations, and bulk selection parameters. Our results show a striking degree of correlation between the genotyping output of both methods, which suggests that the benefit of SFP genotyping in context of BSA can be had with the cheaper, more efficient SNP arrays. As a final proof of concept, we hybridized the DNA from bulks of an F2 mapping population of a Sulfur and Selenium ionomics mutant to both the Arabidopsis ATTILE1R and ATSNPTILE arrays, which produced almost identical results. We have produced R scripts that prompt the user for the required parameters and perform the BSA analysis using the ATSNPTILE1 array and have provided them as supplemental data files.

  7. Regulation of nucleotide excision repair by nuclear lamin b1.

    Directory of Open Access Journals (Sweden)

    Veronika Butin-Israeli

    Full Text Available The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1 in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

  8. Single nucleotide polymorphisms of Kit gene in Chinese indigenous horses.

    Science.gov (United States)

    Han, Haoyuan; Mao, Chunchun; Chen, Ningbo; Lan, Xianyong; Chen, Hong; Lei, Chuzhao; Dang, Ruihua

    2016-02-01

    Kit gene is a genetic determinant of horse white coat color which has been a highly valued trait in horses for at least 2,000 years. Single nucleotide polymorphisms (SNPs) in Kit are of importance due to their strong associations with melanoblast survival during embryonic development. In this study, a mutation analysis of all 21 Kit exons in 14 Chinese domestic horse breeds revealed six SNPs (g.91214T>G, g.143245T>G, g.164297C>T, g.170189C>T, g.171356C>G, and g.171471G>A), which located in 5'-UTR region, intron 6, exon 15, exon 20, intron 20, and exon 21 of the equine Kit gene, respectively. Subsequently, these six SNPs loci were genotyped in 632 Chinese horses by PCR-RFLP or direct sequencing. The six SNPs together defined 18 haplotypes, demonstrating abundant haplotype diversities in Chinese horses. All the mutant alleles and haplotypes were shared among different breeds. But fewer mutations were detected in horses from China than that from abroad, indicating that Chinese horses belong to a more ancient genetic pool. This study will provide fundamental genetic information for evaluating the genetic diversity of Kit gene in Chinese indigenous horse breeds. PMID:27348891

  9. Single Nucleotide Polymorphism Clustering in Systemic Autoimmune Diseases.

    Science.gov (United States)

    Charlon, Thomas; Martínez-Bueno, Manuel; Bossini-Castillo, Lara; Carmona, F David; Di Cara, Alessandro; Wojcik, Jérôme; Voloshynovskiy, Sviatoslav; Martín, Javier; Alarcón-Riquelme, Marta E

    2016-01-01

    Systemic Autoimmune Diseases, a group of chronic inflammatory conditions, have variable symptoms and difficult diagnosis. In order to reclassify them based on genetic markers rather than clinical criteria, we performed clustering of Single Nucleotide Polymorphisms. However naive approaches tend to group patients primarily by their geographic origin. To reduce this "ancestry signal", we developed SNPClust, a method to select large sources of ancestry-independent genetic variations from all variations detected by Principal Component Analysis. Applied to a Systemic Lupus Erythematosus case control dataset, SNPClust successfully reduced the ancestry signal. Results were compared with association studies between the cases and controls without or with reference population stratification correction methods. SNPClust amplified the disease discriminating signal and the ratio of significant associations outside the HLA locus was greater compared to population stratification correction methods. SNPClust will enable the use of ancestry-independent genetic information in the reclassification of Systemic Autoimmune Diseases. SNPClust is available as an R package and demonstrated on the public Human Genome Diversity Project dataset at https://github.com/ThomasChln/snpclust. PMID:27490238

  10. Adenine nucleotides as allosteric effectors of PEA seed glutamine synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, P.J.; Knight, T.J.

    1986-05-01

    The energy charge in the plant cell has been proposed as a regulator of glutamine synthetase (GS) activity. The authors have shown that 2.1 moles of ..gamma..(/sup 32/P)-ATP were bound/mole subunits of purified pea seed GS during complete inactivation with methionine sulfoximine. Since GS has one active site per subunit, the second binding site provides the potential for allosteric regulation of GS by adenine nucleotides. The authors have investigated the inhibition of the ATP-dependent synthetic activity by ADP and AMP. ADP and AMP cannot completely inhibit GS; but ATP does overcome the inhibition by ADP and AMP as shown by plots of % inhibition vs inhibitor concentration. This indicates that inhibition of GS by ADP or AMP is not completely due to competitive inhibition. In the absence of ADP or AMP, double reciprocal plots for ATP are linear below 10 mM; however, in the presence of either ADP or AMP these pots are curvilinear downwards. The ratio of Vm/asymptote is less than 1. The Hill number for ATP in the absence of ADP or AMP is 0.93 but decreases with increasing ADP or AMP to a value of 0.28 with 10 mM ADP. These data are consistent with negative cooperativity by ADP and AMP. Thus, as the ADP/ATP or AMP/ATP ratios are increased GS activity decreases. This is consistent with regulation of GS activity by energy charge in planta.

  11. Single nucleotide polymorphism discovery in barley using autoSNPdb.

    Science.gov (United States)

    Duran, Chris; Appleby, Nikki; Vardy, Megan; Imelfort, Michael; Edwards, David; Batley, Jacqueline

    2009-05-01

    Molecular markers are used to provide the link between genotype and phenotype, for the production of molecular genetic maps and to assess genetic diversity within and between related species. Single nucleotide polymorphisms (SNPs) are the most abundant molecular genetic marker. SNPs can be identified in silico, but care must be taken to ensure that the identified SNPs reflect true genetic variation and are not a result of errors associated with DNA sequencing. The SNP detection method autoSNP has been developed to identify SNPs from sequence data for any species. Confidence in the predicted SNPs is based on sequence redundancy, and haplotype co-segregation scores are calculated for a further independent measure of confidence. We have extended the autoSNP method to produce autoSNPdb, which integrates SNP and gene annotation information with a graphical viewer. We have applied this software to public barley expressed sequences, and the resulting database is available over the Internet. SNPs can be viewed and searched by sequence, functional annotation or predicted synteny with a reference genome, in this case rice. The correlation between SNPs and barley cultivar, expressed tissue type and development stage has been collated for ease of exploration. An average of one SNP per 240 bp was identified, with SNPs more prevalent in the 5' regions and simple sequence repeat (SSR) flanking sequences. Overall, autoSNPdb can provide a wealth of genetic polymorphism information for any species for which sequence data are available. PMID:19386041

  12. MGMT expression: insights into its regulation. 2. Single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Iatsyshyna A. P.

    2013-09-01

    Full Text Available High intra- and interindividual variations in the expression levels of the human O6-methylguanine-DNA methyltransferase (MGMT gene have been observed. This DNA repair enzyme can be a cause of resistance of cancer cells to alkylating chemotherapy. It has been studied the association of single nucleotide polymorphisms (SNPs of MGMT with the risk for different types of cancer, progression-free survival in patients with cancer treated with alkylating chemotherapy, as well as an effect of SNPs on the MGMT gene expression and activity of the enzyme. SNPs have been suggested to be the factors which influence the levels of interindividual variability of the MGMT expression. Therefore, the aim of this paper was to review the experimental data on SNPs of the human MGMT gene, which are associated with cancer, as well as on location of MGMT-SNPs in regulatory and protein-coding regions of the gene in relation to its regulation. Lots of MGMT SNPs, which could affect the gene expression and result in interindividual MGMT variability or the enzyme resistance to pseudosubstrate inhibitors, have been re- vealed within the promoter and enhancer regions, the 5'- and 3'-UTRs and introns of the MGMT gene, as well as within the protein-coding region. Many of them may have regulatory effect.

  13. RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

    OpenAIRE

    Rychlewski Leszek; Bujnicki Janusz M

    2002-01-01

    Abstract Background Escherichia coli guanine-N2 (m2G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain. Results Using a combination of sequence database searches and fold recognition methods it has been demo...

  14. Complete nucleotide sequence of a monopartite Begomovirus and associated satellites infecting Carica papaya in Nepal.

    Science.gov (United States)

    Shahid, M S; Yoshida, S; Khatri-Chhetri, G B; Briddon, R W; Natsuaki, K T

    2013-06-01

    Carica papaya (papaya) is a fruit crop that is cultivated mostly in kitchen gardens throughout Nepal. Leaf samples of C. papaya plants with leaf curling, vein darkening, vein thickening, and a reduction in leaf size were collected from a garden in Darai village, Rampur, Nepal in 2010. Full-length clones of a monopartite Begomovirus, a betasatellite and an alphasatellite were isolated. The complete nucleotide sequence of the Begomovirus showed the arrangement of genes typical of Old World begomoviruses with the highest nucleotide sequence identity (>99 %) to an isolate of Ageratum yellow vein virus (AYVV), confirming it as an isolate of AYVV. The complete nucleotide sequence of betasatellite showed greater than 89 % nucleotide sequence identity to an isolate of Tomato leaf curl Java betasatellite originating from Indonesian. The sequence of the alphasatellite displayed 92 % nucleotide sequence identity to Sida yellow vein China alphasatellite. This is the first identification of these components in Nepal and the first time they have been identified in papaya.

  15. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    LENUS (Irish Health Repository)

    Krause-Gruszczynska, Malgorzata

    2011-12-28

    Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  16. Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Huidong; Eoff, Robert L.; Kozekov, Ivan D.; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter; (Vanderbilt)

    2009-08-13

    Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N{sup 2},N{sup 2}-dimethyl (Me{sub 2})-substituted guanine (N{sup 2},N{sup 2}-Me{sub 2}G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N{sup 2},N{sup 2}-Me{sub 2}G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N{sup 2},N{sup 2}-Me{sub 2}G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N{sup 2},N{sup 2}-Me{sub 2}G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3{prime}-terminal dideoxycytosine (Cdd) opposite template N{sup 2},N{sup 2}-Me{sub 2}G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N{sup 2},N{sup 2}-Me{sub 2}G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N{sup 2},N{sup 2}-Me{sub 2}G, with a second water-mediated hydrogen bond between the N-3 atom of C{sub dd} and the O-6 atom of N{sup 2},N{sup 2}-Me{sub 2}G. A crystal structure of Dpo4 with dTTP opposite template N{sup 2},N{sup 2}-Me{sub 2}G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N{sup 2},N{sup 2}-Me{sub 2}G compared with mono-substituted N{sup 2}-alkyl G adducts.

  17. Effects of hypobaric hypoxia on adenine nucleotide pools, adenine nucleotide transporter activity and protein expression in rat liver

    Institute of Scientific and Technical Information of China (English)

    Cong-Yang Li; Jun-Ze Liu; Li-Ping Wu; Bing Li; Li-Fen Chen

    2006-01-01

    AIM: To explore the effect of hypobaric hypoxia on mitochondrial energy metabolism in rat liver.METHODS: Adult male Wistar rats were exposed to a hypobaric chamber simulating 5000 m high altitude for 23 h every day for 0 (HO), 1 (H1), 5 (HS), 15 (H15) and 30 d (H30) respectively. Rats were sacrificed by decapitation and liver was removed. Liver mitochondria were isolated by differential centrifugation program. The size of adenine nucleotide pool (ATP, ADP, and AMP) in tissue and mitochondria was separated and measured by high performance liquid chromatography (HPLC). The adenine nucleotide transporter (ANT) activity was determined by isotopic technique. The ANT total protein level was determined by Western blot. RESULTS: Compared with HO group, intra-mitochondrial ATP content decreased in all hypoxia groups. However,the H5 group reached the lowest point (70.6%) (P< 0.01)when compared to the control group. Intra-mitochondrial ADP and AMP level showed similar change in all hypoxia groups and were significantly lower than that in HO group. In addition, extra-mitochondrial ATP and ADP content decreased significantly in all hypoxia groups.Furthermore, extra-mitochondrial AMP in groups H5, H15and H30 was significantly lower than that in HO group,whereas H1 group had no marked change compared to the control situation. The activity of ANT in hypoxia groups decreased significantly, which was the lowest in H5 group (55.7%) (P<0.01) when compared to HO group. ANT activity in H30 group was higher than in H15 group, but still lower than that in HO group. ANT protein level in H5, H15, H30 groups, compared with HO group decreased significantly, which in H5 group was the lowest, being 27.1% of that in HO group (P<0.01). ANT protein level in H30 group was higher than in H15 group,but still lower than in HO group.CONCLUSION: Hypobaric hypoxia decreases the mitochondrial ATP content in rat liver, while mitochondrial ATP level recovers during long-term hypoxia exposure.The lower

  18. Actinides and rare earths complexation with adenosine phosphate nucleotides

    International Nuclear Information System (INIS)

    Organophosphorus compounds are important molecules in both nuclear industry and living systems fields. Indeed, several extractants of organophosphorus compounds (such as TBP, HDEHP) are used in the nuclear fuel cycle reprocessing and in the biological field. For instance, the nucleotides are organophosphates which play a very important role in various metabolic processes. Although the literature on the interactions of actinides with inorganic phosphate is abundant, published studies with organophosphate compounds are generally limited to macroscopic and / or physiological approaches. The objective of this thesis is to study the structure of several organophosphorus compounds with actinides to reach a better understanding and develop new specific buildings blocks. The family of the chosen molecules for this procedure consists of three adenine nucleotides mono, bi and triphosphate (AMP, adenosine monophosphate - ADP, adenosine diphosphate - ATP, adenosine triphosphate) and an amino-alkylphosphate (AEP O-phosphoryl-ethanolamine). Complexes synthesis was conducted in aqueous and weakly acidic medium (2.8-4) for several lanthanides (III) (Lu, Yb, Eu) and actinides (U (VI), Th (IV) and Am (III)). Several analytical and spectroscopic techniques have been used to describe the organization of the synthesized complexes: spectrometric analysis performed by FTIR and NMR were used to identify the functional groups involved in the complexation, analysis by ESI-MS and pH-metric titration were used to determine the solution speciation and EXAFS analyzes were performed on Mars beamline of the SOLEIL synchrotron, have described the local cation environment, for both solution and solid compounds. Some theoretical approaches of DFT were conducted to identify stable structures in purpose of completing the experimental studies. All solid complexes (AMP, ADP, ATP and AEP) have polynuclear structures, while soluble ATP complexes are mononuclear. For all synthesized complexes, it has been

  19. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology

    Institute of Scientific and Technical Information of China (English)

    Sarah C Shuck; Emily A Short; John J Turchi

    2008-01-01

    Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

  20. Resistance issues with new nucleoside/nucleotide backbone options.

    Science.gov (United States)

    Wainberg, Mark A; Turner, Dan

    2004-09-01

    The nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) remain an enduring feature of combination therapy. As NRTI/NtRTI options continue to expand, questions arise about how best to combine these agents to create effective dual NRTI/NtRTI backbones in antiretroviral regimens while avoiding treatment-emergent drug resistance. Clinicians must consider how NRTIs/NtRTIs such as tenofovir DF (TDF), abacavir (ABC), and emtricitabine (FTC), as well as new once-daily and coformulated NRTIs/NtRTIs, interact with older agents when combined in novel regimens and how sequencing the new NRTIs can preserve future treatment options. Resistance data from clinical trials have revealed important information on the patterns, prevalence, and effects of resistance seen among patients experiencing virologic failure. In recent years, the prevalence of some mutations such as M184V and Q151M has remained relatively constant, while the L74V mutation, the 69 insertions, and thymidine analogue mutations have decreased in prevalence. Other mutations such as K65R and Y115F, while still relatively uncommon, are increasing in prevalence. This increase may be due to the use of new treatment combinations that select for these mutations at a higher rate. Clinical trials suggest that new regimens containing TDF or ABC select for K65R and that this mutation is observed more frequently with TDF; in contrast, L74V is observed more frequently in ABC-containing regimens but is not commonly selected by TDF-containing regimens. Several lines of evidence are converging to suggest that the presence of zidovudine may decrease the risk of L74V and K65R in ABC- or TDF-containing regimens. This review summarizes the clinical implications of resistance profiles associated with new NRTI/NtRTI regimens in current use and in advanced clinical studies. PMID:15319668

  1. Cortisol promotes endoplasmic glucose production via pyridine nucleotide redox.

    Science.gov (United States)

    Wang, Zengmin; Mick, Gail J; Xie, Rongrong; Wang, Xudong; Xie, Xuemei; Li, Guimei; McCormick, Kenneth L

    2016-04-01

    Both increased adrenal and peripheral cortisol production, the latter governed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), contribute to the maintenance of fasting blood glucose. In the endoplasmic reticulum (ER), the pyridine nucleotide redox state (NADP/NADPH) is dictated by the concentration of glucose-6-phosphate (G6P) and the coordinated activities of two enzymes, hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. However, luminal G6P may similarly serve as a substrate for hepatic glucose-6-phophatase (G6Pase). A tacit belief is that the G6P pool in the ER is equally accessible to both H6PDH and G6Pase. Based on our inhibition studies and kinetic analysis in isolated rat liver microsomes, these two aforesaid luminal enzymes do share the G6P pool in the ER, but not equally. Based on the kinetic modeling of G6P flux, the ER transporter for G6P (T1) preferentially delivers this substrate to G6Pase; hence, the luminal enzymes do not share G6P equally. Moreover, cortisol, acting through 11β-HSD1, begets a more reduced pyridine redox ratio. By altering this luminal redox ratio, G6P flux through H6PDH is restrained, allowing more G6P for the competing enzyme G6Pase. And, at low G6P concentrations in the ER lumen, which occur during fasting, this acute cortisol-induced redox adjustment promotes glucose production. This reproducible cortisol-driven mechanism has been heretofore unrecognized. PMID:26860459

  2. Network analysis of single nucleotide polymorphisms in asthma

    Science.gov (United States)

    Renkonen, Jutta; Joenväärä, Sakari; Parviainen, Ville; Mattila, Pirkko; Renkonen, Risto

    2010-01-01

    Background: Asthma is a chronic inflammatory disease of the airways with a complex genetic background. In this study, we carried out a meta-analysis of single nucleotide polymorphisms (SNPs) thought to be associated with asthma. Methods: The literature (PubMed) was searched for SNPs within genes relevant in asthma. The SNP-modified genes were converted to corresponding proteins, and their protein–protein interactions were searched from six different databases. This interaction network was analyzed using annotated vocabularies (ontologies), such as the Gene Ontology and Nature pathway interaction databases. Results: In total, 127 genes with SNPs related to asthma were found in the literature. The corresponding proteins were then entered into a large protein–protein interaction network with the help of various databases. Ninety-six SNP-related proteins had more than one interacting protein each, and a network containing 309 proteins and 644 connections was generated. This network was significantly enriched with a gene ontology entitled “protein binding” and several of its daughter categories, including receptor binding and cytokine binding, when compared with the background human proteome. In the detailed analysis, the chemokine network, including eight proteins and 13 toll-like receptors, were shown to interact with each other. Of great interest are the nonsynonymous SNPs which code for an alternative amino acid sequence of proteins and, of the toll-like receptor network, TLR1, TLR4, TLR5, TLR6, TLR10, IL4R, and IL13 are among these. Conclusions: Protein binding, toll-like receptors, and chemokines dominated in the asthma-related protein interaction network. Systems level analysis of allergy-related mutations can provide new insights into the pathogenetic mechanisms of disease. PMID:21437052

  3. Nucleotide sequence and temporal expression of a baculovirus regulatory gene.

    Science.gov (United States)

    Guarino, L A; Summers, M D

    1987-07-01

    The nucleotide sequence of a trans-activating regulatory gene (IE-1) of the baculovirus Autographa californica nuclear polyhedrosis virus has been determined. This gene encodes a protein of 581 amino acids with a predicted molecular weight of 66,856. A DNA fragment containing the entire coding sequence of IE-1 was inserted downstream of an RNA promoter. Subsequent cell-free transcription and translation directed the synthesis of a single peptide with an apparent molecular weight of 70,000. Quantitative S1 nuclease analysis indicated that IE-1 was maximally synthesized during a 1-h virus adsorption period and that steady-state levels of IE-1 message were maintained during the first 24 h of infection. Northern blot hybridization indicated that several late transcripts which overlap the IE-1 gene were transcribed from both strands. The precise locations of the 5' and 3' ends of these overlapping transcripts were mapped using S1 nuclease. The overlapping transcripts were grouped in two transcriptional units. One unit was composed of IE-1 and overlapping gamma transcripts which initiated upstream of IE-1 and terminated downstream of IE-1. The other unit, transcribed from the opposite strand, consisted of gamma transcripts with coterminal 5' ends and extended 3' ends. The shorter, more abundant transcripts in this unit overlapped 30 to 40 bases of IE-1 at the 3' end, while the longer transcripts overlapped the entire IE-1 gene. Transcription of several early A. californica nuclear polyhedrosis virus genes, in addition to 39K, was shown to be trans-activated by IE-1, indicating that IE-1 may have a central role in the regulation of beta-gene expression. PMID:16789264

  4. ACG: rapid inference of population history from recombining nucleotide sequences

    Directory of Open Access Journals (Sweden)

    O'Fallon Brendan D

    2013-02-01

    Full Text Available Abstract Background Reconstruction of population history from genetic data often requires Monte Carlo integration over the genealogy of the samples. Among tools that perform such computations, few are able to consider genetic histories including recombination events, precluding their use on most alignments of nuclear DNA. Explicit consideration of recombinations requires modeling the history of the sequences with an Ancestral Recombination Graph (ARG in place of a simple tree, which presents significant computational challenges. Results ACG is an extensible desktop application that uses a Bayesian Markov chain Monte Carlo procedure to estimate the posterior likelihood of an evolutionary model conditional on an alignment of genetic data. The ancestry of the sequences is represented by an ARG, which is estimated from the data with other model parameters. Importantly, ACG computes the full, Felsenstein likelihood of the ARG, not a pairwise or composite likelihood. Several strategies are used to speed computations, and ACG is roughly 100x faster than a similar, recombination-aware program. Conclusions Modeling the ancestry of the sequences with an ARG allows ACG to estimate the evolutionary history of recombining nucleotide sequences. ACG can accurately estimate the posterior distribution of population parameters such as the (scaled population size and recombination rate, as well as many aspects of the recombinant history, including the positions of recombination breakpoints, the distribution of time to most recent common ancestor along the sequence, and the non-recombining trees at individual sites. Multiple substitution models and population size models are provided. ACG also provides a richly informative graphical interface that allows users to view the evolution of model parameters and likelihoods in real time.

  5. Sequencing genes in silico using single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Zhang Xinyi

    2012-01-01

    Full Text Available Abstract Background The advent of high throughput sequencing technology has enabled the 1000 Genomes Project Pilot 3 to generate complete sequence data for more than 906 genes and 8,140 exons representing 697 subjects. The 1000 Genomes database provides a critical opportunity for further interpreting disease associations with single nucleotide polymorphisms (SNPs discovered from genetic association studies. Currently, direct sequencing of candidate genes or regions on a large number of subjects remains both cost- and time-prohibitive. Results To accelerate the translation from discovery to functional studies, we propose an in silico gene sequencing method (ISS, which predicts phased sequences of intragenic regions, using SNPs. The key underlying idea of our method is to infer diploid sequences (a pair of phased sequences/alleles at every functional locus utilizing the deep sequencing data from the 1000 Genomes Project and SNP data from the HapMap Project, and to build prediction models using flanking SNPs. Using this method, we have developed a database of prediction models for 611 known genes. Sequence prediction accuracy for these genes is 96.26% on average (ranges 79%-100%. This database of prediction models can be enhanced and scaled up to include new genes as the 1000 Genomes Project sequences additional genes on additional individuals. Applying our predictive model for the KCNJ11 gene to the Wellcome Trust Case Control Consortium (WTCCC Type 2 diabetes cohort, we demonstrate how the prediction of phased sequences inferred from GWAS SNP genotype data can be used to facilitate interpretation and identify a probable functional mechanism such as protein changes. Conclusions Prior to the general availability of routine sequencing of all subjects, the ISS method proposed here provides a time- and cost-effective approach to broadening the characterization of disease associated SNPs and regions, and facilitating the prioritization of candidate

  6. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    Science.gov (United States)

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  7. Old concepts, new molecules and current approaches applied to the bacterial nucleotide signalling field

    Science.gov (United States)

    2016-01-01

    Signalling nucleotides are key molecules that help bacteria to rapidly coordinate cellular pathways and adapt to changes in their environment. During the past 10 years, the nucleotide signalling field has seen much excitement, as several new signalling nucleotides have been discovered in both eukaryotic and bacterial cells. The fields have since advanced quickly, aided by the development of important tools such as the synthesis of modified nucleotides, which, combined with sensitive mass spectrometry methods, allowed for the rapid identification of specific receptor proteins along with other novel genome-wide screening methods. In this review, we describe the principle concepts of nucleotide signalling networks and summarize the recent work that led to the discovery of the novel signalling nucleotides. We also highlight current approaches applied to the research in the field as well as resources and methodological advances aiding in a rapid identification of nucleotide-specific receptor proteins. This article is part of the themed issue ‘The new bacteriology’. PMID:27672152

  8. Electrostatics of DNA nucleotide-carbon nanotube hybrids evaluated from QM:MM simulations.

    Science.gov (United States)

    Chehel Amirani, Morteza; Tang, Tian

    2015-12-14

    Biomolecule-functionalized carbon nanotubes (CNTs) have been studied vastly in recent years due to their potential applications for instance in cancer detection, purification and separation of CNTs, and nanoelectronics. Studying the electrostatic potential generated by a biomolecule-CNT hybrid is important in predicting its interactions with the surrounding environment such as charged particles and surfaces. In this paper, we performed atomistic simulations using a QM:MM approach to evaluate the electrostatic potential and charge transfer for a hybrid structure formed by a DNA nucleotide and a CNT in solution. Four types of DNA nucleotides and two CNTs with chiralities of (4,4) and (7,0) were considered. The types of nucleotides and CNTs were both found to play important roles in the electrostatic potential and charge transfer of the hybrid. At the same distance from the CNT axis, the electrostatic potential for the nucleotide-(4,4) CNT hybrids was found to be stronger compared with that for the nucleotide-(7,0) CNT hybrids. Higher electric charge was also shown to be transferred from the DNA nucleotides to the (7,0) CNT compared with the (4,4) CNT. These results correlate with the previous finding that the nucleotides bound more tightly to the (7,0) CNT compared with the (4,4) CNT. PMID:26542447

  9. The study of neighboring nucleotide composition and transition/transversion bias

    Institute of Scientific and Technical Information of China (English)

    ZHAO Hui; LI Qizhai; LI Jun; ZENG Changqing; HU Songnian; YU Jun

    2006-01-01

    Base substitution is one of the raw fuels that produce genetic variation and drive evolution. Recent studies have shown that the genome components affect mutation patterns to some extent. In order to infer the correlation between the Transition/Transversion ratio (Ts/Tv) and the number of immediately adjacent A&T nucleotides, we investigated 3611007 Oryza sativa SNPs (including 45462 coding SNPs, and 242811 intronic SNPs) and 32019 Arabidopsis SNPs. The results show that Ts/Tv is negatively correlated with the number of immediately adjacent A&T in O. sativa and Arabidopsis. We further calculated AT2 (the number of SNPs whose immediately adjacent nucleotides are either A or T) and AT0 (the number of SNPs whose immediately adjacent nucleotides are either C or G) for all 6 types of SNPs. C/G SNP of O. sativa and Arabidopsis has the highest AT2/AT0, which denotes C/G SNP may be influenced by the adjacent A&T nucleotides mostly. For SNPs in O. sativa, the neighboring effect of A&T nucleotides is limited to 2 nucleotides on both sides; for SNPs in Arabidopsis, the effect extends no more than 4 nucleotides on both sides.

  10. Dietary Nucleotides Supplementation and Liver Injury in Alcohol-Treated Rats: A Metabolomics Investigation

    Directory of Open Access Journals (Sweden)

    Xiaxia Cai

    2016-03-01

    Full Text Available Background: Previous studies suggested that nucleotides were beneficial for liver function, lipid metabolism and so on. The present study aimed to investigate the metabolic response of dietary nucleotides supplementation in alcohol-induced liver injury rats. Methods: Five groups of male Wistar rats were used: normal control group (basal diet, equivalent distilled water, alcohol control group (basal diet, 50% alcohol (v/v, dextrose control group (basal diet, isocaloric amount of dextrose, and 0.04% and 0.16% nucleotides groups (basal diet supplemented with 0.4 g and 1.6 g nucleotides kg−1 respectively, 50% alcohol (v/v. The liver injury was measured through traditional liver enzymes, expression of oxidative stress markers and histopathological examination. Ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF-MS was applied to identify liver metabolite profiles. Results: Nucleotides supplementation prevented the progression of hepatocyte steatosis. The levels of total proteins, globulin, alanine aminotransferase, aspartate aminotransferase, total cholesterol triglyceride, as well as the oxidative stress markers altered by alcohol, were improved by nucleotides supplementation. Elevated levels of liver bile acids (glycocholic acid, chenodeoxyglycocholic acid, and taurodeoxycholic acid, as well as lipids (stearic acid, palmitic acid, oleic acid, phosphatidylcholine, and lysophosphatidylethanolamine in alcohol-treated rats were reversed by nucleotides supplementation. In addition, supplementation with nucleotides could increase the levels of amino acids, including valyl-Leucine, l-leucine, alanyl-leucine and l-phenylalanine. Conclusion: These data indicate potential biomarkers and confirm the benefit of dietary nucleotides on alcoholic liver injury.

  11. Effects of six anaesthetic agents on UDP-glucuronic acid and other nucleotides in rat liver.

    Science.gov (United States)

    Christensson, P I; Eriksson, G

    1985-08-01

    Anaesthesia affects the liver nucleotide pool. It was the aim of the present study to examine how anaesthesia for 60 min with pentobarbital, ketamin + diazepam, halothane, enflurane and isoflurane may influence the nucleotide pool in the rat liver, studied with isotachophoresis. It was found that none of the agents gave both safe and reproducible anaesthesia without affecting the nucleotide pools or affecting the experiments in some other way. Halothane and isoflurane were the two best alternatives with respect to both efficiency and safety. Isoflurane may be preferable since it gives a higher energy charge.

  12. The nucleotide sequence of the uvrD gene of E. coli.

    OpenAIRE

    Finch, P W; Emmerson, P T

    1984-01-01

    The nucleotide sequence of a cloned section of the E. coli chromosome containing the uvrD gene has been determined. The coding region for the UvrD protein consists of 2,160 nucleotides which would direct the synthesis of a polypeptide 720 amino acids long with a calculated molecular weight of 82 kd. The predicted amino acid sequence of the UvrD protein has been compared with the amino acid sequences of other known adenine nucleotide binding proteins and a common sequence has been identified, ...

  13. Predicting protein-binding RNA nucleotides using the feature-based removal of data redundancy and the interaction propensity of nucleotide triplets.

    Science.gov (United States)

    Choi, Sungwook; Han, Kyungsook

    2013-11-01

    Several learning approaches have been used to predict RNA-binding amino acids in a protein sequence, but there has been little attempt to predict protein-binding nucleotides in an RNA sequence. One of the reasons is that the differences between nucleotides in their interaction propensity are much smaller than those between amino acids. Another reason is that RNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding RNA nucleotides is much harder than predicting RNA-binding amino acids. We developed a new method that removes data redundancy in a training set of sequences based on their features. The new method constructs a larger and more informative training set than the standard redundancy removal method based on sequence similarity, and the constructed dataset is guaranteed to be redundancy-free. We computed the interaction propensity (IP) of nucleotide triplets by applying a new definition of IP to an extensive dataset of protein-RNA complexes, and developed a support vector machine (SVM) model to predict protein binding sites in RNA sequences. In a 5-fold cross-validation with 812 RNA sequences, the SVM model predicted protein-binding nucleotides with an accuracy of 86.4%, an F-measure of 84.8%, and a Matthews correlation coefficient of 0.66. With an independent dataset of 56 RNA sequences that were not used in training, the resulting accuracy was 68.1% with an F-measure of 71.7% and a Matthews correlation coefficient of 0.35. To the best of our knowledge, this is the first attempt to predict protein-binding RNA nucleotides in a given RNA sequence from the sequence data alone. The SVM model and datasets are freely available for academics at http://bclab.inha.ac.kr/primer.

  14. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Science.gov (United States)

    2010-07-01

    ... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data...

  15. Myosin individualized: single nucleotide polymorphisms in energy transduction

    Directory of Open Access Journals (Sweden)

    Wieben Eric D

    2010-03-01

    Full Text Available Abstract Background Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC. Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism. Results An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain. Conclusions Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these

  16. Database of amino acid-nucleotide contacts in the DNA complexes with homeodomain family proteins

    International Nuclear Information System (INIS)

    The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires analysis of the physicochemical characteristics of these contacts, of the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used for comparison and classification of interfaces of the protein-DNA complexes

  17. Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids.

    Science.gov (United States)

    Gissot, Arnaud; Camplo, Michel; Grinstaff, Mark W; Barthélémy, Philippe

    2008-04-21

    Amphiphilic molecules based on nucleosides, nucleotides and oligonucleotides are finding more and more biotechnological applications. This Perspective highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology.

  18. Unraveling the cellular context of cyclic nucleotide signaling proteins by chemical proteomics

    NARCIS (Netherlands)

    Corradini, E.

    2015-01-01

    Understanding the molecular mechanisms which regulate signal transduction is fundamental to the development of therapeutic molecules for the treatment of several diseases. In particular, signaling proteins, such as cyclic nucleotide dependent enzymes are the orchestrators of many tissue functions. A

  19. A Nucleotide Signature for the Identification of American Ginseng and Its Products

    Directory of Open Access Journals (Sweden)

    Yang eLiu

    2016-03-01

    Full Text Available American ginseng (derived from Panax quinquefolius is one of the most widely used medicinal herbs in the world. Because of its high price and increasing demand, there are many adulterants on the market. The proposed internal transcribed spacer 2 (ITS2 has been used to identify raw medicinal materials, but it is not suitable for the identification of Chinese patent medicine ingredients. Therefore, a short barcode for the identification of processed American ginseng and its corresponding Chinese patent medicines would be profitable. In this study, 94 samples of American ginseng and Asian ginseng were collected from all over the world. The internal transcribed spacer 2 (ITS2 region was sequenced, and a nucleotide signature was developed based on one single nucleotide polymorphism (SNP site unique to American ginseng. The nucleotide signature (atcactcctt tgcgggagtc gaggcgg consists of 27 bases over the length of the ITS2 sequence (420 bp. Furthermore, we also designed primer pairs to amplify the nucleotide signature; the specific primer pair 4F/4R has been found to be unique to the ginseng species and capable of amplifying the nucleotide signatures from Chinese patent medicines and decoctions. We used the nucleotide signature method to inspect ginseng products in Chinese patent medicines; 24 batches of Chinese patent medicine from stores in Beijing were amplified and sequenced successfully. Using the double peaks at the SNP sites of the nucleotide signature, 5 batches were found to be counterfeits, and 2 batches were found to contain adulterants. Thus, this nucleotide signature, with only 27 bp, has broadened the application of DNA barcoding in identification of decoctions, Chinese patent medicines and other ginseng products with degraded DNA. This method can rapidly identify ginseng products and could also be developed as an on-site detection method.

  20. Efficient reverse transcription using locked nucleic acid nucleotides towards the evolution of nuclease resistant RNA aptamers

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Edwards, Stacey L;

    2012-01-01

    We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers.......We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers....

  1. Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

    Energy Technology Data Exchange (ETDEWEB)

    Leng, Q.; Mercier, R.W.; Yao, W.; Berkowitz, G.A.

    1999-11-01

    Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMO or cGMO binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e., demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. The authors report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the {alpha}-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K{sup +} uptake system complements growth inhibition only when lipophilic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus lawvis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K{sup +} currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca{sup 2+} only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence identifying a plant member of this ion channel family.

  2. A comprehensive study of ribozyme-mediated nucleotide and sugar chemistries

    OpenAIRE

    Lau, Matthew

    2009-01-01

    The ability of RNA catalysts (ribozymes) to synthesize nucleotides as basic building blocks for the assembly of larger RNA polymers is an important aspect of the 'RNA World' hypothesis. To examine the ability of RNAs to catalyze this chemistry, we performed an in vitro selection in my first project and successfully isolated ribozymes capable of synthesizing a purine nucleotide (6-thioguanosine, 6SG) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine (6SGua). Compared to t...

  3. Targeting one carbon metabolism with an antimetabolite disrupts pyrimidine homeostasis and induces nucleotide overflow

    OpenAIRE

    Ser, Zheng; GAO, XIA; Johnson, Christelle; Mehrmohamadi, Mahya; Liu, Xiaojing; Li, SiQi; Locasale, Jason W.

    2016-01-01

    Anti-metabolite agents that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used anti-metabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of ...

  4. Nucleoside-nucleotide free diet protects rat colonic mucosa from damage induced by trinitrobenzene sulphonic acid.

    OpenAIRE

    Adjei, A A; Morioka, T.; Ameho, C K; Yamauchi, K.; Kulkarni, A. D.; Al-Mansouri, H M; Kawajiri, A; Yamamoto, S.

    1996-01-01

    BACKGROUND: Growing evidence suggests that intestinal recovery from injury induced by radiation, endotoxin, and protein deficiency is improved by the ingestion of nucleosides and nucleotides. AIM: This study examined the effect of dietary nucleosides and nucleotides supplementation on trinitrobenzene sulphonic acid induced colonic damage in experimental colitis. METHODS: Sprague-Dawley rats were randomised into two groups and fed nucleic acid free 20% casein diet (control) or this diet supple...

  5. Nucleotide Diversity and Selection Signature in the Domesticated Silkworm, Bombyx mori, and Wild Silkworm, Bombyx mandarina

    OpenAIRE

    Guo, Yi; Shen, Yi-Hong; Sun, Wei; Kishino, Hirohisa; Xiang, Zhong-Huai; Zhang, Ze

    2011-01-01

    To investigate the patterns of nucleotide diversity in domesticated silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) and its wild relative, Chinese wild silkworm, Bombyx mandarina Moore, we sequenced nine nuclear genes. Neutrality test and coalescent simulation for these genes were performed to look at bottleneck intensity and selection signature; linkage disequilibrium (LD) within and between loci was employed to investigate allele association. As a result, B. mori lost 33–49% of nucleotid...

  6. Acid-base properties of functionalised tripodal polyamines and their interaction with nucleotides and nucleic acids.

    Science.gov (United States)

    Sornosa-Ten, Alejandra; Albelda, M Teresa; Frías, Juan C; García-España, Enrique; Llinares, José M; Budimir, Ana; Piantanida, Ivo

    2010-06-01

    Novel, highly positively charged tripodal polyamines with appended heterocyclic moieties revealed an intriguing panel of protonation species within the biologically relevant range. Studied compounds bind nucleotide monophosphates by mostly electrostatic interactions but only the imidazole analogue showed selectivity toward UMP in respect to other nucleotides. Strong binding of all the studied compounds to both ds-DNA and ds-RNA is to some extent selective toward the latter, showing rather rare RNA over DNA preference. PMID:20485792

  7. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water

    OpenAIRE

    Cafferty, Brian J.; Fialho, David M.; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V.

    2016-01-01

    The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ...

  8. Tailing and 3'-end labeling of RNA with yeast poly(A) polymerase and various nucleotides.

    OpenAIRE

    Martin, G; Keller, W

    1998-01-01

    We have tested conditions for the labeling and tailing the 3'-end of RNAs with yeast poly(A) polymerase. Conditions were optimized for addition of NTP, dNTP, or ddNTP nucleotides to RNA. ATP, GTP, and UTP were useful for adding homopolymer tracts of various lengths. The nonradioactive nucleotides biotin-N6-ATP and digoxigenin-11-UTP also were used efficiently.

  9. Nucleotide Dependent Intrinsic Fluorescence Changes of W29 and W36 in Smooth Muscle Myosin

    OpenAIRE

    van Duffelen, Marilyn; Chrin, Lynn R.; Berger, Christopher L.

    2004-01-01

    The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogen...

  10. Biochemistry of an olfactory purinergic system: dephosphorylation of excitatory nucleotides and uptake of adenosine

    Energy Technology Data Exchange (ETDEWEB)

    Trapido-Rosenthal, H.G.; Carr, W.E.; Gleeson, R.A.

    1987-10-01

    The olfactory organ of the spiny lobster, Panulirus argus, is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of (/sup 3/H)-adenosine is saturable with increasing concentration, linear with time for up to 3 h, sodium dependent, insensitive to moderate pH changes and has a Km of 7.1 microM and a Vmax of 5.2 fmol/sensillum/min (573 fmol/micrograms of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; /sup 3/H from adenine-labeled AMP or ATP is internalized, whereas 32P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; /sup 3/H from AMP is internalized at the same rate as /sup 3/H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog alpha, beta-methylene ADP. Collectively, these results indicate that the enzymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.

  11. Organization of Nucleotides in Different Environments and the Formation of Pre-Polymers

    Science.gov (United States)

    Himbert, Sebastian; Chapman, Mindy; Deamer, David W.; Rheinstädter, Maikel C.

    2016-08-01

    RNA is a linear polymer of nucleotides linked by a ribose-phosphate backbone. Polymerization of nucleotides occurs in a condensation reaction in which phosphodiester bonds are formed. However, in the absence of enzymes and metabolism there has been no obvious way for RNA-like molecules to be produced and then encapsulated in cellular compartments. We investigated 5‧-adenosine monophosphate (AMP) and 5‧-uridine monophosphate (UMP) molecules confined in multi-lamellar phospholipid bilayers, nanoscopic films, ammonium chloride salt crystals and Montmorillonite clay, previously proposed to promote polymerization. X-ray diffraction was used to determine whether such conditions imposed a degree of order on the nucleotides. Two nucleotide signals were observed in all matrices, one corresponding to a nearest neighbour distance of 4.6 Å attributed to nucleotides that form a disordered, glassy structure. A second, smaller distance of 3.4 Å agrees well with the distance between stacked base pairs in the RNA backbone, and was assigned to the formation of pre-polymers, i.e., the organization of nucleotides into stacks of about 10 monomers. Such ordering can provide conditions that promote the nonenzymatic polymerization of RNA strands under prebiotic conditions. Experiments were modeled by Monte-Carlo simulations, which provide details of the molecular structure of these pre-polymers.

  12. Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs.

    Science.gov (United States)

    Hacker, Stephan M; Buntz, Annette; Zumbusch, Andreas; Marx, Andreas

    2015-11-20

    Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells. PMID:26274552

  13. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides.

    Science.gov (United States)

    Lehninger, A L; Vercesi, A; Bababunmi, E A

    1978-04-01

    Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca(2+) and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca(2+) is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as beta-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca(2+). Successive cycles of Ca(2+) release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca(2+)-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca(2+), mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport. PMID:25436

  14. Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

    Science.gov (United States)

    Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun

    2015-11-10

    Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure. PMID:26279371

  15. Neighboring-Nucleotide Effects on the Mutation Patterns of the Rice Genome

    Institute of Scientific and Technical Information of China (English)

    Hui Zhao; Qi-Zhai Li; Chang-Qing Zeng; Huan-Ming Yang; Jun Yu

    2005-01-01

    DNA composition dynamics across genomes of diverse taxonomy is a major subject of genome analyses. DNA composition changes are characteristics of both replication and repair machineries. We investigated 3,611,007 single nucleotide polymorphisms (SNPs) generated by comparing two sequenced rice genomes from distant inbred lines (subspecies), including those from 242,811 introns and 45,462 protein-coding sequences (CDSs). Neighboring-nucleotide effects (NNEs) of these SNPs are diverse, depending on structural content-based classifications (genomewide, intronic, and CDS) and sequence context-based categories (A/C, A/G, A/T,C/G, C/T, and G/T substitutions) of the analyzed SNPs. Strong and evident NNEs and nucleotide proportion biases surrounding the analyzed SNPs were observed in 1-3 bp sequences on both sides of an SNP. Strong biases were observed around neighboring nucleotides of protein-coding SNPs, which exhibit a periodicity of three in nucleotide content, constrained by a combined effect of codon-related rules and DNA repair mechanisms. Unlike a previous finding in the human genome,we found negative correlation between GC contents of chromosomes and the magnitude of corresponding bias of nucleotide C at -1 site and G at +1 site. These results will further our understanding of the mutation mechanism in rice as well as its evolutionary implications.

  16. Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs.

    Science.gov (United States)

    Hacker, Stephan M; Buntz, Annette; Zumbusch, Andreas; Marx, Andreas

    2015-11-20

    Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

  17. Genome-wide patterns of recombination, linkage disequilibrium and nucleotide diversity from pooled resequencing and single nucleotide polymorphism genotyping unlock the evolutionary history of Eucalyptus grandis.

    Science.gov (United States)

    Silva-Junior, Orzenil B; Grattapaglia, Dario

    2015-11-01

    We used high-density single nucleotide polymorphism (SNP) data and whole-genome pooled resequencing to examine the landscape of population recombination (ρ) and nucleotide diversity (ϴw ), assess the extent of linkage disequilibrium (r(2) ) and build the highest density linkage maps for Eucalyptus. At the genome-wide level, linkage disequilibrium (LD) decayed within c. 4-6 kb, slower than previously reported from candidate gene studies, but showing considerable variation from absence to complete LD up to 50 kb. A sharp decrease in the estimate of ρ was seen when going from short to genome-wide inter-SNP distances, highlighting the dependence of this parameter on the scale of observation adopted. Recombination was correlated with nucleotide diversity, gene density and distance from the centromere, with hotspots of recombination enriched for genes involved in chemical reactions and pathways of the normal metabolic processes. The high nucleotide diversity (ϴw = 0.022) of E. grandis revealed that mutation is more important than recombination in shaping its genomic diversity (ρ/ϴw = 0.645). Chromosome-wide ancestral recombination graphs allowed us to date the split of E. grandis (1.7-4.8 million yr ago) and identify a scenario for the recent demographic history of the species. Our results have considerable practical importance to Genome Wide Association Studies (GWAS), while indicating bright prospects for genomic prediction of complex phenotypes in eucalypt breeding.

  18. Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons

    Directory of Open Access Journals (Sweden)

    Robert N. Azad

    2015-05-01

    Full Text Available We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D2. Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996 J. Am. Chem. Soc. 118, 7929–7940. Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL.

  19. Association of Toll-like receptor 2 Arg753Gln and Toll-like receptor 1 Ile602Ser single-nucleotide polymorphisms with leptospirosis in an Argentine population.

    Science.gov (United States)

    Cédola, Maia; Chiani, Yosena; Pretre, Gabriela; Alberdi, Lucrecia; Vanasco, Bibiana; Gómez, Ricardo M

    2015-06-01

    Toll-like receptor 2 (TLR2), a member of the Toll-like receptor family, plays an important role in the recognition of and subsequent immune response activation against leptospirosis in humans. The genetic polymorphism in TLR2 of an arginine to glutamine substitution at residue 753 (Arg753Gln) has been associated with a negative influence on TLR2 function, which may, in turn, determine the innate host response to Leptospira spp. This bacterium signals through TLR2/TLR1 heterodimers in human cells. The aim of the present study was to investigate the Arg753Gln single-nucleotide polymorphism (SNP) of the TLR2 gene, and the isoleucine to serine transversion at position 602 (Ile602Ser) of the TLR1 gene (previously associated with Lyme disease), in leptospirosis patients compared to healthy controls, carrying out a retrospective case/control study. The TLR2 polymorphism adenine (A) allele was observed in 7.3% of leptospirosis patients but was not found in the control group, whereas the guanine (G) allele of the TLR1 polymorphism was found in 63.6% of patients and 41.6% of controls. Susceptibility to leptospirosis disease was increased 10.57-fold for carriers of the TLR2 G/A genotype (P=0.0493) and 3.85-fold for carriers of the TLR1 G/G genotype (P=0.0428). Furthermore, the risk of developing hepatic insufficiency and jaundice was increased 18.86- and 27.60-fold for TLR2 G/A carriers, respectively. Similarly, the risk of developing jaundice was increased 12.67-fold for TLR1 G allele carriers (G/G and T/G genotypes). In conclusion, the present data suggest that the TLR2 Arg753Gln and TLR1 Ile602Ser SNPs influence the risk of developing leptospirosis and its severity.

  20. Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

    Science.gov (United States)

    Wang, Yijin; Wang, Wenshi; Xu, Lei; Zhou, Xinying; Shokrollahi, Ehsan; Felczak, Krzysztof; van der Laan, Luc J W; Pankiewicz, Krzysztof W; Sprengers, Dave; Raat, Nicolaas J H; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2016-05-01

    Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway

  1. Electrostatics of DNA nucleotide-carbon nanotube hybrids evaluated from QM:MM simulations

    Science.gov (United States)

    Chehel Amirani, Morteza; Tang, Tian

    2015-11-01

    Biomolecule-functionalized carbon nanotubes (CNTs) have been studied vastly in recent years due to their potential applications for instance in cancer detection, purification and separation of CNTs, and nanoelectronics. Studying the electrostatic potential generated by a biomolecule-CNT hybrid is important in predicting its interactions with the surrounding environment such as charged particles and surfaces. In this paper, we performed atomistic simulations using a QM:MM approach to evaluate the electrostatic potential and charge transfer for a hybrid structure formed by a DNA nucleotide and a CNT in solution. Four types of DNA nucleotides and two CNTs with chiralities of (4,4) and (7,0) were considered. The types of nucleotides and CNTs were both found to play important roles in the electrostatic potential and charge transfer of the hybrid. At the same distance from the CNT axis, the electrostatic potential for the nucleotide-(4,4) CNT hybrids was found to be stronger compared with that for the nucleotide-(7,0) CNT hybrids. Higher electric charge was also shown to be transferred from the DNA nucleotides to the (7,0) CNT compared with the (4,4) CNT. These results correlate with the previous finding that the nucleotides bound more tightly to the (7,0) CNT compared with the (4,4) CNT.Biomolecule-functionalized carbon nanotubes (CNTs) have been studied vastly in recent years due to their potential applications for instance in cancer detection, purification and separation of CNTs, and nanoelectronics. Studying the electrostatic potential generated by a biomolecule-CNT hybrid is important in predicting its interactions with the surrounding environment such as charged particles and surfaces. In this paper, we performed atomistic simulations using a QM:MM approach to evaluate the electrostatic potential and charge transfer for a hybrid structure formed by a DNA nucleotide and a CNT in solution. Four types of DNA nucleotides and two CNTs with chiralities of (4,4) and (7

  2. Multifactor dimensionality reduction analysis identifies specific nucleotide patterns promoting genetic polymorphisms

    Directory of Open Access Journals (Sweden)

    Arehart Eric

    2009-03-01

    Full Text Available Abstract Background The fidelity of DNA replication serves as the nidus for both genetic evolution and genomic instability fostering disease. Single nucleotide polymorphisms (SNPs constitute greater than 80% of the genetic variation between individuals. A new theory regarding DNA replication fidelity has emerged in which selectivity is governed by base-pair geometry through interactions between the selected nucleotide, the complementary strand, and the polymerase active site. We hypothesize that specific nucleotide combinations in the flanking regions of SNP fragments are associated with mutation. Results We modeled the relationship between DNA sequence and observed polymorphisms using the novel multifactor dimensionality reduction (MDR approach. MDR was originally developed to detect synergistic interactions between multiple SNPs that are predictive of disease susceptibility. We initially assembled data from the Broad Institute as a pilot test for the hypothesis that flanking region patterns associate with mutagenesis (n = 2194. We then confirmed and expanded our inquiry with human SNPs within coding regions and their flanking sequences collected from the National Center for Biotechnology Information (NCBI database (n = 29967 and a control set of sequences (coding region not associated with SNP sites randomly selected from the NCBI database (n = 29967. We discovered seven flanking region pattern associations in the Broad dataset which reached a minimum significance level of p ≤ 0.05. Significant models (p Conclusion The present study represents the first use of this computational methodology for modeling nonlinear patterns in molecular genetics. MDR was able to identify distinct nucleotide patterning around sites of mutations dependent upon the observed nucleotide change. We discovered one flanking region set that included five nucleotides clustered around a specific type of SNP site. Based on the strongly associated patterns identified in

  3. Intramolecular interactions in aminoacyl nucleotides: Implications regarding the origin of genetic coding and protein synthesis

    Science.gov (United States)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Watkins, C. L.; Hall, L. M.

    1986-01-01

    Cellular organisms store information as sequences of nucleotides in double stranded DNA. This information is useless unless it can be converted into the active molecular species, protein. This is done in contemporary creatures first by transcription of one strand to give a complementary strand of mRNA. The sequence of nucleotides is then translated into a specific sequence of amino acids in a protein. Translation is made possible by a genetic coding system in which a sequence of three nucleotides codes for a specific amino acid. The origin and evolution of any chemical system can be understood through elucidation of the properties of the chemical entities which make up the system. There is an underlying logic to the coding system revealed by a correlation of the hydrophobicities of amino acids and their anticodonic nucleotides (i.e., the complement of the codon). Its importance lies in the fact that every amino acid going into protein synthesis must first be activated. This is universally accomplished with ATP. Past studies have concentrated on the chemistry of the adenylates, but more recently we have found, through the use of NMR, that we can observe intramolecular interactions even at low concentrations, between amino acid side chains and nucleotide base rings in these adenylates. The use of this type of compound thus affords a novel way of elucidating the manner in which amino acids and nucleotides interact with each other. In aqueous solution, when a hydrophobic amino acid is attached to the most hydrophobic nucleotide, AMP, a hydrophobic interaction takes place between the amino acid side chain and the adenine ring. The studies to be reported concern these hydrophobic interactions.

  4. RNA:(guanine-N2 methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

    Directory of Open Access Journals (Sweden)

    Rychlewski Leszek

    2002-04-01

    Full Text Available Abstract Background Escherichia coli guanine-N2 (m2G methyltransferases (MTases RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain. Results Using a combination of sequence database searches and fold recognition methods it has been demonstrated that the N-termini of RsmC and RsmD are related to each other and that they represent a "degenerated" version of the C-terminal MTase domain. Novel members of the YbiN family from Archaea and Eukaryota were also indentified. It is inferred that YbiN and both domains of RsmC and RsmD are closely related to a family of putative MTases from Gram-positive bacteria and Archaea, typified by the Mj0882 protein from M. jannaschii (1dus in PDB. Based on the results of sequence analysis and structure prediction, the residues involved in cofactor binding, target recognition and catalysis were identified, and the mechanism of the guanine-N2 methyltransfer reaction was proposed. Conclusions Using the known Mj0882 structure, a comprehensive analysis of sequence-structure-function relationships in the family of genuine and putative m2G MTases was performed. The results provide novel insight into the mechanism of m2G methylation and will serve as a platform for experimental analysis of numerous uncharacterized N-MTases.

  5. Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry.

    Science.gov (United States)

    Ito, Jun; Herter, Thomas; Baidoo, Edward E K; Lao, Jeemeng; Vega-Sánchez, Miguel E; Michelle Smith-Moritz, A; Adams, Paul D; Keasling, Jay D; Usadel, Björn; Petzold, Christopher J; Heazlewood, Joshua L

    2014-03-01

    Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in their cell wall sugar compositions and indicated significant differences in the levels of 6 out of 12 nucleotide sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery rates with minimal matrix effects. The approach results in a significant improvement in sensitivity when applied to plant samples over currently employed techniques.

  6. Nucleotide precursors prevent folic acid-resistant neural tube defects in the mouse.

    Science.gov (United States)

    Leung, Kit-Yi; De Castro, Sandra C P; Savery, Dawn; Copp, Andrew J; Greene, Nicholas D E

    2013-09-01

    Closure of the neural tube during embryogenesis is a crucial step in development of the central nervous system. Failure of this process results in neural tube defects, including spina bifida and anencephaly, which are among the most common birth defects worldwide. Maternal use of folic acid supplements reduces risk of neural tube defects but a proportion of cases are not preventable. Folic acid is thought to act through folate one-carbon metabolism, which transfers one-carbon units for methylation reactions and nucleotide biosynthesis. Hence suboptimal performance of the intervening reactions could limit the efficacy of folic acid. We hypothesized that direct supplementation with nucleotides, downstream of folate metabolism, has the potential to support neural tube closure. Therefore, in a mouse model that exhibits folic acid-resistant neural tube defects, we tested the effect of specific combinations of pyrimidine and purine nucleotide precursors and observed a significant protective effect. Labelling in whole embryo culture showed that nucleotides are taken up by the neurulating embryo and incorporated into genomic DNA. Furthermore, the mitotic index was elevated in neural folds and hindgut of treated embryos, consistent with a proposed mechanism of neural tube defect prevention through stimulation of cellular proliferation. These findings may provide an impetus for future investigations of supplemental nucleotides as a means to prevent a greater proportion of human neural tube defects than can be achieved by folic acid alone.

  7. Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

    Science.gov (United States)

    Kulkarni, A D; Fanslow, W C; Drath, D B; Rudolph, F B; Van Buren, C T

    1986-02-01

    Although enzyme defects in purine metabolism have revealed the importance of these substrates to maintenance of a normal immune response, the role of exogenous nucleotides on the cells that mediate the host defense system has remained largely unexplored. Recent investigations have revealed that dietary nucleotides are vital to the maintenance of cell-mediated responses to antigen stimulation. To test the influence of dietary nucleotide deprivation on resistance to infection, Balb/c mice were maintained on chow, a nucleotide-free (NF) diet, or an NF diet repleted with adenine, uracil, or RNA. Mice on the NF diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus, while chow-fed and RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the NF diet had decreased phagocytic activity as measured by uptake of radiolabeled bacteria compared with mice maintained on the NF diet supplemented with adenine, uracil, or RNA. No change in S aureus antibody response was noted on the various diets. Although the mechanism of this suppression of nonspecific immunity remains unclear, provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge. PMID:3947217

  8. Comprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing.

    Science.gov (United States)

    Rutherford, Steven T; Valastyan, Julie S; Taillefumier, Thibaud; Wingreen, Ned S; Bassler, Bonnie L

    2015-11-01

    Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of ∼ 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.

  9. Identification of Single Nucleotide Polymorphism on Growth Hormone Gene in Aceh Cattle

    Directory of Open Access Journals (Sweden)

    E. M. Sari

    2013-04-01

    Full Text Available This research was aimed to identify the changes of nucleotide (Single Nucleotide Polymorphism growth hormone gene in the population of Aceh cattle. There were 44 samples of DNA sequenced, and a few samples from Gen Bank (M57764. Based on the analysis using MEGA program, it was identified one new mutation on exon five on 2230 bp in which C nucleotide turned into T nucleotide, and this was called Silent Mutation (Leusine–Leusine/ CTC–CTT. The frequency of Single Nucleotide Polymorphism (SNP genotype on 2230 bp (C/T was CC (0.36, TT (0.14 and CT (0.50. The genotype TT was not possessed by Aceh cattle from Saree, but possessed by those from Banda Aceh and Indrapuri. Chi-square test showed not significant differences in allele frequencies for three population. The frequency of genotype SNP on 2291 bp (A/C was AC (0.11 and CC (0.89. The frequency of allele C was higher than allele A and T.

  10. Sublingual Nucleotides Prolong Run Time to Exhaustion in Young Physically Active Men

    Directory of Open Access Journals (Sweden)

    Sergej M. Ostojic

    2013-11-01

    Full Text Available Although dietary nucleotides have been determined to be required for normal immune function, there is limited direct interventional evidence confirming performance-enhancing effects of sublingual nucleotides in humans. A double-blind, placebo-controlled, randomized trial was conducted to evaluate the effect of sublingual nucleotides (50 mg/day administered for 14 days in thirty young healthy physically active males, on endurance performance and immune responses. Fasting white blood cell count, natural killer cells (NKC number, NKC cytotoxic activity, and serum immunoglobulin (IgA, IgM, IgG, and time to exhaustion, peak rate of perceived exertion, peak heart rate, and peak running speed during the exercise test were measured at baseline (day 0 and post-intervention (day 14. Time to exhaustion, as well as serum immunoglobulin A and NKC cytotoxic activity, were significantly higher at day 14 (p < 0.05 in participants supplemented with nucleotides compared with those who consumed placebo. No significant differences in other parameters were observed between groups at post-intervention. No volunteers withdrew before the end of the study nor reported any vexatious side effects of supplementation. The results of the present study suggest that sublingual nucleotides may provide pertinent benefit as both an ergogenic and immunostimulatory additive in active males.

  11. Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2).

    Science.gov (United States)

    Chung, Christina Z; Jo, David Hyung Suk; Heinemann, Ilka U

    2016-08-01

    The nontemplated addition of single or multiple nucleotides to RNA transcripts is an efficient means to control RNA stability and processing. Cytoplasmic RNA adenylation and the less well-known uridylation are post-transcriptional mechanisms regulating RNA maturation, activity, and degradation. Gld2 is a member of the noncanonical poly(A) polymerases, which include enzymes with varying nucleotide specificity, ranging from strictly ATP to ambiguous to exclusive UTP adding enzymes. Human Gld2 has been associated with transcript stabilizing miRNA monoadenylation and cytoplasmic mRNA polyadenylation. Most recent data have uncovered an unexpected miRNA uridylation activity, which promotes miRNA maturation. These conflicting data raise the question of Gld2 nucleotide specificity. Here, we biochemically characterized human Gld2 and demonstrated that it is a bona fide adenylyltransferase with only weak activity toward other nucleotides. Despite its sequence similarity with uridylyltransferases (TUT4, TUT7), Gld2 displays an 83-fold preference of ATP over UTP. Gld2 is a promiscuous enzyme, with activity toward miRNA, pre-miRNA, and polyadenylated RNA substrates. Apo-Gld2 activity is restricted to adding single nucleotides and processivity likely relies on additional RNA-binding proteins. A phylogeny of the PAP/TUTase superfamily suggests that uridylyltransferases, which are derived from distinct adenylyltransferase ancestors, arose multiple times during evolution via insertion of an active site histidine. A corresponding histidine insertion into the Gld2 active site alters substrate specificity from ATP to UTP. PMID:27284165

  12. On the role of a coumarin derivative for sensing applications: Nucleotide identification using a micellar system.

    Science.gov (United States)

    Bettoschi, Alexandre; Ceglie, Andrea; Lopez, Francesco; Meli, Valeria; Murgia, Sergio; Tamburro, Manuela; Caltagirone, Claudia; Cuomo, Francesca

    2016-09-01

    The recognition of nucleotides is of crucial importance because they are the basic constituents of nucleic acids. The present study is focused on the selective interaction between a novel amphiphilic fluorophore containing coumarin and imidazole, CI (1-methyl-3-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-imidazol-3-ium bromide), and different nucleotide-monophosphates (NMPs). It was supposed that the solubilization of the low water soluble CI in a micelle system of hexadecyltrimethylammonium chloride (CTAC) would make the coumarin moiety of CI available to the interaction with the water-soluble NMPs. Changes in CTAC critical micelle concentration suggested that CI strongly interacted with the host cationic surfactant, thus forming a positively charged interface enriched with coumarin able to interact with the anionic NMPs. Steady-state fluorescence quenching revealed that CI/CTAC system was capable of distinguish between purine- and pyrimidine-based nucleotides. A modified Stern-Volmer equation permitted the use of a quenching model that accounted for the possible interactions between the micelles and the nucleotides. The data analysis allowed calculating selective parameters that differentiated according to the type of nucleotide either at 25 or 50°C. Our results established the utility of the novel coumarin derivative fluorophore, supported by the simple and suitable micellar systems, as a tool for DNA sensing applications.

  13. On the role of a coumarin derivative for sensing applications: Nucleotide identification using a micellar system.

    Science.gov (United States)

    Bettoschi, Alexandre; Ceglie, Andrea; Lopez, Francesco; Meli, Valeria; Murgia, Sergio; Tamburro, Manuela; Caltagirone, Claudia; Cuomo, Francesca

    2016-09-01

    The recognition of nucleotides is of crucial importance because they are the basic constituents of nucleic acids. The present study is focused on the selective interaction between a novel amphiphilic fluorophore containing coumarin and imidazole, CI (1-methyl-3-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-imidazol-3-ium bromide), and different nucleotide-monophosphates (NMPs). It was supposed that the solubilization of the low water soluble CI in a micelle system of hexadecyltrimethylammonium chloride (CTAC) would make the coumarin moiety of CI available to the interaction with the water-soluble NMPs. Changes in CTAC critical micelle concentration suggested that CI strongly interacted with the host cationic surfactant, thus forming a positively charged interface enriched with coumarin able to interact with the anionic NMPs. Steady-state fluorescence quenching revealed that CI/CTAC system was capable of distinguish between purine- and pyrimidine-based nucleotides. A modified Stern-Volmer equation permitted the use of a quenching model that accounted for the possible interactions between the micelles and the nucleotides. The data analysis allowed calculating selective parameters that differentiated according to the type of nucleotide either at 25 or 50°C. Our results established the utility of the novel coumarin derivative fluorophore, supported by the simple and suitable micellar systems, as a tool for DNA sensing applications. PMID:27236839

  14. Complete nucleotide sequences of two isolates ofCherry virus A from sweet cherry in China

    Institute of Scientific and Technical Information of China (English)

    GAO Rui; LI Shi-fang; LU Mei-guang

    2016-01-01

    Cherry virus A(CVA) is a member of the genusCapilovirus, in the familyBetalfexiviridae. The infection rate of CVA was high in sweet cherry in China. We determined the complete nucleotide sequences of two isolates of CVA from Tai’an, Shan-dong Province, China using high ifdelity PCR enzymes and speciifc primer pairs for amplifying long fragments in RT-PCR and RACE. The ful-length sequences from isolates ChTA11 and ChTA12 are both 7382 nucleotide (nt) long, excluding the poly(A) tail, encode two open reading frames (ORFs) and have similar genome organization to the two isolates in Gen-Bank. The complete nucleotide sequence of ChTA11 is 98.2 and 81.2% nt identity to the isolates from Germany and India in GenBank, respectively, and the ChTA12 isolate is 98.2 and 81.0% similar. Analysis of the nucleotide and amino acid sequences showed that the domain of unknown function (DUF1717) is more variable compared with other domains. This is the ifrst report of the complete nucleotide sequences of CVA isolates infecting sweet cherry in China.

  15. Structural basis for dual nucleotide selectivity of aminoglycoside 2''-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases.

    Science.gov (United States)

    Shi, Kun; Berghuis, Albert M

    2012-04-13

    Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.

  16. Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B).

    Science.gov (United States)

    de Araujo, Elvin D; Alvarez, Claudia P; López-Alonso, Jorge P; Sooklal, Clarissa R; Stagljar, Marijana; Kanelis, Voula

    2015-09-11

    The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels. PMID:26198630

  17. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Science.gov (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  18. Complete nucleotide sequence of the new potexvirus "Alstroemeria virus X". Brief report.

    Science.gov (United States)

    Fuji, S; Shinoda, K; Ikeda, M; Furuya, H; Naito, H; Fukumoto, F

    2005-11-01

    A flexuous virus was isolated in Japan from an alstroemeria plant showing mosaic symptoms. The virus had a broad host range but had systemically latent infectivity in alstroemeria. The virus was assigned to the genus Potexvirus based on morphology and physical properties and on an analysis of the complete nucleotide sequence. The genomic RNA of the virus was 7,009 nucleotides in length, excluding the 3'-terminal poly (A) tail. It contained five open reading frames (ORFs), which was consistent with other members of the genus Potexvirus. Although nucleotide sequences of the ORFs differ from previously reported potexviruses, a phylogenetic analysis placed it phylogenetically close to Narcissus mosaic virus and Scallion virus X. Therefore, we propose that this virus should be designated as Alstroemeria virus X (AlsVX). PMID:15986173

  19. Crystal structure of cyclic nucleotide-binding-like protein from Brucella abortus.

    Science.gov (United States)

    He, Zheng; Gao, Yuan; Dong, Jing; Ke, Yuehua; Li, Xuemei; Chen, Zeliang; Zhang, Xuejun C

    2015-12-25

    The cyclic nucleotide-binding (CNB)-like protein (CNB-L) from Brucella abortus shares sequence homology with CNB domain-containing proteins. We determined the crystal structure of CNB-L at 2.0 Å resolution in the absence of its C-terminal helix and nucleotide. The 3D structure of CNB-L is in a two-fold symmetric form. Each protomer shows high structure similarity to that of cGMP-binding domain-containing proteins, and likely mimics their nucleotide-free conformation. A key residue, Glu17, mediates the dimerization and prevents binding of cNMP to the canonical ligand-pocket. The structurally observed dimer of CNB-L is stable in solution, and thus is likely to be biologically relevant.

  20. Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

    Science.gov (United States)

    Trucco, Verónica; de Breuil, Soledad; Bejerman, Nicolás; Lenardon, Sergio; Giolitti, Fabián

    2014-06-01

    The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host. PMID:24510307