Sample records for camp-regulated guanine nucleotide

  1. Dbl family guanine nucleotide exchange factors. (United States)

    Zheng, Y


    The Dbl family of guanine nucleotide exchange factors are multifunctional molecules that transduce diverse intracellular signals leading to the activation of Rho GTPases. The tandem Dbl-homology and pleckstrin-homology domains shared by all members of this family represent the structural module responsible for catalyzing the GDP-GTP exchange reaction of Rho proteins. Recent progress in genomic, genetic, structural and biochemical studies has implicated Dbl family members in diverse biological processes, including growth and development, skeletal muscle formation, neuronal axon guidance and tissue organization. The detailed pictures of their autoregulation, agonist-controlled activation and mechanism of interaction with Rho GTPase substrates, have begun to emerge.

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

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    B Josh Lane


    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.

  3. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases (United States)

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


    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.

  4. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases (United States)

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


    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

  5. A Minimal Rac Activation Domain in the Unconventional Guanine Nucleotide Exchange Factor Dock180†


    Xin WU; Ramachandran, Sekar; Cerione, Richard A.; Erickson, Jon W.


    Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR...

  6. A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange


    Rossman, Kent L.; Worthylake, David K.; Snyder, Jason T; Siderovski, David P.; Campbell, Sharon L; Sondek, John


    Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain ...

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

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    Chen Wei


    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.

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


    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

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

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    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.

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

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    Claire J Fenech


    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.

  11. Guanine nucleotide binding protein-like 3 is a potential prognosis indicator of gastric cancer. (United States)

    Chen, Jing; Dong, Shuang; Hu, Jiangfeng; Duan, Bensong; Yao, Jian; Zhang, Ruiyun; Zhou, Hongmei; Sheng, Haihui; Gao, Hengjun; Li, Shunlong; Zhang, Xianwen


    Guanine nucleotide binding protein-like 3 (GNL3) is a GIP-binding nuclear protein that has been reported to be involved in various biological processes, including cell proliferation, cellular senescence and tumorigenesis. This study aimed to investigate the expression level of GNL3 in gastric cancer and to evaluate the relationship between its expression and clinical variables and overall survival of gastric cancer patients. The expression level of GNL3 was examined in 89 human gastric cancer samples using immunohistochemistry (IHC) staining. GNL3 in gastric cancer tissues was significantly upregulated compared with paracancerous tissues. GNL3 expression in adjacent non-cancerous tissues was associated with sex and tumor size. Survival analyses showed that GNL3 expression in both gastric cancer and adjacent non-cancerous tissues were not related to overall survival. However, in the subgroup of patients with larger tumor size (≥ 6 cm), a close association was found between GNL3 expression in gastric cancer tissues and overall survival. GNL3-positive patients had a shorter survival than GNL3-negative patients. Our study suggests that GNL3 might play an important role in the progression of gastric cancer and serve as a biomarker for poor prognosis in gastric cancer patients.

  12. Characterization of leukemia-associated Rho guanine nucleotide exchange factor (LARG) expression during murine development. (United States)

    Becknell, Brian; Shen, Tiansheng; Maghraby, Eman; Taya, Shinichiro; Kaibuchi, Kozo; Caligiuri, Michael A; Marcucci, Guido


    LARG (leukemia-associated Rho guanine nucleotide exchange factor, ARHGEF12) was originally identified as a fusion partner of the MLL gene at 11q23 in human acute myeloid leukemia. We have previously demonstrated that the LARG protein activates RhoA, a member of the Rho family of small GTPases, by catalyzing the exchange of GTP for GDP. Experiments in Drosophila melanogaster have implicated RhoA and its regulators in a spectrum of developmental processes-including gastrulation, neurite outgrowth, and epidermal morphogenesis; however, the role of these genes during mammalian development is incompletely understood. Herein, we investigate the expression of the murine LARG homologue during embryogenesis and in adult animals, by a combination of mRNA in situ hybridization and immunohistochemical detection of the LARG protein. We observe that LARG transcript and protein are undetectable prior to embryonic day 14. Beginning at this stage, LARG is expressed in the skin, intestinal epithelium, and smooth muscle layers of the intestine, bronchi, and vasculature. This specific distribution is maintained at later stages of development and into adulthood. Finally, we demonstrate colocalization of the LARG protein with the insulin-like growth factor-I (IGF-1) receptor, suggesting a potential physiologic role for LARG as an activator of RhoA in response to IGF-1.

  13. Disruption of oligomerization induces nucleocytoplasmic shuttling of leukemia-associated rho Guanine-nucleotide exchange factor. (United States)

    Grabocka, Elda; Wedegaertner, Philip B


    The rgsRhoGEFs comprise a subfamily of three guanine nucleotide exchange factors, which function in linking heterotrimeric G-proteins to the monomeric RhoGTPase. Here, we reveal the novel finding that oligomerization of leukemia-associated RhoGEF (LARG) functions to prevent nucleocytoplasmic shuttling and to retain LARG in the cytoplasm. We establish that oligomerization is mediated by a predicted coiled-coil sequence (amino acids 1507-1520) in the extreme C terminus of LARG and that substitution of isoleucines 1507/1510 with alanines disrupts homo-oligomerization and leads to nucleocytoplasmic shuttling via the CRM1 nuclear transport pathway. In addition, we demonstrate that induced dimerization of an otherwise nuclear monomeric LARG mutant promotes cytoplasmic localization. Furthermore, we establish that nuclear import of monomeric LARG is mediated by the nuclear localization sequence (29)PTDKKQK(35) in the extreme N terminus. We propose that nucleocytoplasmic shuttling provides a mechanism for spatially regulating the activity of LARG toward its cytoplasmic targets and potentially new nuclear targets.

  14. How not to do kinetics: examples involving GTPases and guanine nucleotide exchange factors. (United States)

    Goody, Roger S


    Guanine nucleotide exchange factors (GEFs) are crucial regulators of the action of GTPases in signal transduction and cellular regulation. Although their basic mechanism of action has been apparent for almost 20 years, there are still misconceptions concerning their properties, and these are confounded by superficial or incorrect interpretation of experimental results in individual cases. Here, an example is described in which an incorrect mechanism was derived because of an inadequate analysis of kinetic results. In a second example, a case is discussed where certain GTP analogs were erroneously described as being able to function as low molecular mass GEFs. In both cases, a lack of distinction between rates, rate constants, and apparent rate constants, together with a disregard of relative signal amplitudes, led to the misinterpretations. In a final example, it is shown how the lack of an appropriate kinetic investigation led to the false conclusion that a secreted protein from Legionella pneumophila can act not only as a GEF towards eukaryotic Rab1 but also as a factor that is able to actively dissociate the stable complex between Rab1 and GDP dissociation inhibitor.

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

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    Fluharty Eric


    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.

  16. The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin (United States)

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


    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

  17. Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases


    Lee, Chan-Soo; Choi, Chang-Ki; Shin, Eun-Young; Schwartz, Martin Alexander; Kim, Eung-Gook


    Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology–pleckstrin homology modu...

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

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


    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...

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

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    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.

  20. Haploinsufficiency of the Sec7 guanine nucleotide exchange factor gea1 impairs septation in fission yeast.

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    Alan M Eckler

    Full Text Available Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs. Activated Arfs recruit coat protein complex 1 (COP-I to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/-, the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/- strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/- cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/- mutant strain. Importantly, gea1+/- cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/- cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

  1. Small-molecule inhibitors targeting G-protein-coupled Rho guanine nucleotide exchange factors. (United States)

    Shang, Xun; Marchioni, Fillipo; Evelyn, Chris R; Sipes, Nisha; Zhou, Xuan; Seibel, William; Wortman, Matthew; Zheng, Yi


    The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein-coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG-RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme-substrate pair of GEF-RhoA at their binding interface that leads to enhanced efficacy and specificity.

  2. Catching Functional Modes and Structural Communication in Dbl Family Rho Guanine Nucleotide Exchange Factors. (United States)

    Raimondi, Francesco; Felline, Angelo; Fanelli, Francesca


    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.

  3. Trp(56) of rac1 specifies interaction with a subset of guanine nucleotide exchange factors. (United States)

    Gao, Y; Xing, J; Streuli, M; Leto, T L; Zheng, Y


    Signaling specificity of Rho GTPase pathways is achieved in part by selective interaction between members of the Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. For example, Trio, GEF-H1, and Tiam1 are a subset of GEFs that specifically activate Rac1 but not the closely related Cdc42. The Rac1 specificity of these GEFs appears to be governed by Rac1-GEF binding interaction. To understand the detailed mechanism underlying the GEF specificity issue, we have analyzed a panel of chimeras made between Rac1 and Cdc42 and examined a series of point mutants of Rac1 made at the switch I, switch II, and beta(2)/beta(3) regions for their ability to interact with and to be activated by the GEFs. The results reveal that Rac1 residues of both the switch I and switch II regions are involved in GEF docking and GEF-mediated nucleotide disruption, because mutation of Asp(38), Asn(39), Gln(61), Tyr(64), or Arg(66)/Leu(67) into Ala results in the loss of GEF binding, whereas mutation at Tyr(32), Asp(65), or Leu(70)/Ser(71) leads to the loss of GEF catalysis while retaining the binding capability. The region between amino acids 53-72 of Rac1 is required for specific recognition and activation by the GEFs, and Trp(56) in beta(3) appears to be the critical determinant. Introduction of Trp(56) to Cdc42 renders it fully responsive to the Rac-specific GEF in vitro and in cells. Further, a polypeptide derived from the beta(3) region of Rac1 including the Trp(56) residue serves as a specific inhibitor for Rac1 interaction with the GEFs. Taken together, these results indicate that Trp(56) is the necessary and sufficient determinant of Rac1 for discrimination by the subset of Rac1-specific GEFs and suggest that a compound mimicking Trp(56) action could be explored as an interfering reagent specifically targeting Rac1 activation.

  4. The Crystal Structure of Cdc42 in Complex with Collybisin II, a Gephyrin-Interacting Guanine Nucleotide Exchange Factor

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    Xiang,S.; Kim, E.; Connelly, J.; Nassar, N.; Kirsch, J.; WinkingSchwartz, G.; Schindelin, H.


    The synaptic localization of ion channel receptors is essential for efficient synaptic transmission and the precise regulation of diverse neuronal functions. In the central nervous system, ion channel receptors reside in the postsynaptic membrane where they are juxtaposed to presynaptic terminals. For proper function, these ion channels have to be anchored to the cytoskeleton, and in the case of the inhibitory glycine and {gamma}-amino-butyric acid type A (GABA{sub A}) receptors this interaction is mediated by a gephyrin centered scaffold. Highlighting its central role in this receptor anchoring scaffold, gephyrin interacts with a number of proteins, including the neurospecific guanine nucleotide exchange factor collybistin. Collybistin belongs to the Dbl family of guanine nucleotide exchange factors, occurs in multiple splice variants, and is specific for Cdc42, a small GTPase belonging to the Rho family. The 2.3 Angstroms resolution crystal structure of the Cdc42--collybistin II complex reveals a novel conformation of the switch I region of Cdc42. It also provides the first direct observation of structural changes in the relative orientation of the Dbl-homology domain and the pleckstrin-homology domain in the same Dbl family protein. Biochemical data indicate that gephyrin negatively regulates collybistin activity.

  5. ARHGEF7 (Beta-PIX acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2.

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    Karina Haebig

    Full Text Available BACKGROUND: Mutations within the leucine-rich repeat kinase 2 (LRRK2 gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. CONCLUSIONS/SIGNIFICANCE: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i a feedback control mechanism for LRRK2 activity as well as (ii an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis.

  6. Conformational change upon ligand binding and dynamics of the PDZ domain from leukemia-associated Rho guanine nucleotide exchange factor. (United States)

    Liu, Jiangxin; Zhang, Jiahai; Yang, Yinshan; Huang, Hongda; Shen, Weiqun; Hu, Qi; Wang, Xingsheng; Wu, Jihui; Shi, Yunyu


    Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in betaB/betaC and betaE/alphaB loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the (15)N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the betaB/betaC and betaE/alphaB loops affects the PDZ-peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding.

  7. Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs

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    Ehrenberg Måns


    Full Text Available Abstract Background During the translation of mRNA into polypeptide, elongation factor G (EF-G catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recent model, EF-G operates like a 'motor protein' and drives translocation of the peptidyl-tRNA after GTP hydrolysis. In both the classical and motor protein models, GDP-to-GTP exchange is assumed to occur spontaneously on 'free' EF-G even in the absence of a guanine-nucleotide exchange factor (GEF. Results We have made a number of findings that challenge both models. First, free EF-G in the cell is likely to be in the GDP-bound form. Second, the ribosome acts as the GEF for EF-G. Third, after guanine-nucleotide exchange, EF-G in the GTP-bound form moves the tRNA2-mRNA complex to an intermediate translocation state in which the mRNA is partially translocated. Fourth, subsequent accommodation of the tRNA2-mRNA complex in the post-translocation state requires GTP hydrolysis. Conclusion These results, in conjunction with previously published cryo-electron microscopy reconstructions of the ribosome in various functional states, suggest a novel mechanism for translocation of tRNAs on the ribosome by EF-G. Our observations suggest that the ribosome is a universal guanosine-nucleotide exchange factor for EF-G as previously shown for the class-II peptide-release factor 3.

  8. Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs (United States)

    Zavialov, Andrey V; Hauryliuk, Vasili V; Ehrenberg, Måns


    Background During the translation of mRNA into polypeptide, elongation factor G (EF-G) catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recent model, EF-G operates like a 'motor protein' and drives translocation of the peptidyl-tRNA after GTP hydrolysis. In both the classical and motor protein models, GDP-to-GTP exchange is assumed to occur spontaneously on 'free' EF-G even in the absence of a guanine-nucleotide exchange factor (GEF). Results We have made a number of findings that challenge both models. First, free EF-G in the cell is likely to be in the GDP-bound form. Second, the ribosome acts as the GEF for EF-G. Third, after guanine-nucleotide exchange, EF-G in the GTP-bound form moves the tRNA2-mRNA complex to an intermediate translocation state in which the mRNA is partially translocated. Fourth, subsequent accommodation of the tRNA2-mRNA complex in the post-translocation state requires GTP hydrolysis. Conclusion These results, in conjunction with previously published cryo-electron microscopy reconstructions of the ribosome in various functional states, suggest a novel mechanism for translocation of tRNAs on the ribosome by EF-G. Our observations suggest that the ribosome is a universal guanosine-nucleotide exchange factor for EF-G as previously shown for the class-II peptide-release factor 3. PMID:15985150

  9. Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.



    mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutati...

  10. Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho. (United States)

    Fukuhara, S; Chikumi, H; Gutkind, J S


    A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.

  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


    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. Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding. (United States)

    Huang, C F; Chuang, N N


    A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form.

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

    Directory of Open Access Journals (Sweden)

    Geldner Niko


    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.

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

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa


    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.

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

    Directory of Open Access Journals (Sweden)

    Andrzej M Woyda-Ploszczyca

    Full Text Available 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. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay. (United States)

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


    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.

  17. Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases. (United States)

    Lee, Chan-Soo; Choi, Chang-Ki; Shin, Eun-Young; Schwartz, Martin Alexander; Kim, Eung-Gook


    Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.

  18. 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.

  19. Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase (United States)

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


    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.

  20. The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling. (United States)

    Beveridge, Ryan D; Staples, Christopher J; Patil, Abhijit A; Myers, Katie N; Maslen, Sarah; Skehel, J Mark; Boulton, Simon J; Collis, Spencer J


    We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders.

  1. Overexpression of GEFT, a Rho family guanine nucleotide exchange factor, predicts poor prognosis in patients with rhabdomyosarcoma. (United States)

    Sun, Chao; Liu, Chunxia; Li, Shugang; Li, Hongan; Wang, Yuanyuan; Xie, Yuwen; Li, Bingcheng; Cui, Xiaobin; Chen, Yunzhao; Zhang, Wenjie; Li, Feng


    Rhabdomyosarcoma (RMS) is one of the most common soft-tissue sarcomas in children and adolescents with poor prognosis. Yet, there is lack of effective prognostic biomarkers for RMS. The present study, therefore, aimed to explore potential biomarkers for RMS based on our previous findings using array comparative genomic hybridization. We investigated guanine nucleotide exchange factor, GEFT, at expression level in 45 RMS patients and 36 normal striated muscle controls using immunohistochemistry using tissue microarrays. The expression rate of GEFT in RMS samples (42/45, 93.33%) was significantly higher (Prate of GEFT in RMS (31/45, 68.89%) was also significantly higher (P<0.05) than that in normal controls (0/36, 0.00%). Increased expression of GEFT correlated significantly with advanced disease stages (stages III/IV) (P=0.001), lymph node metastasis (P=0.019), and distant metastasis (P=0.004), respectively, in RMS patients. In addition, RMS patients having overexpressed GEFT experienced worse overall survival (OS) than those having low levels of GEFT (P=0.001). GEFT overexpression was determined to be an independent prognostic factor for poor OS in RMS patients (hazard ratio: 3.491, 95% confidence interval: 1.121-10.871, P=0.004). In conclusion, these observations provide the first evidence of GEFT overexpression in RMS and its correlations with disease aggressiveness and metastasis. These findings suggest that GEFT may serve as a promising biomarker predicting poor prognosis in RMS patients, thus implying its potential as a therapeutic target.

  2. 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


    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.

  3. Iodide transporter NIS regulates cancer cell motility and invasiveness by interacting with the Rho guanine nucleotide exchange factor LARG. (United States)

    Lacoste, Claire; Hervé, Julie; Bou Nader, Myriam; Dos Santos, Alexandre; Moniaux, Nicolas; Valogne, Yannick; Montjean, Rodrick; Dorseuil, Olivier; Samuel, Didier; Cassio, Doris; Portulano, Carla; Carrasco, Nancy; Bréchot, Christian; Faivre, Jamila


    A number of solute carrier (SLC) proteins are subject to changes in expression and activity during carcinogenesis. Whether these changes play a role in carcinogenesis is unclear, except for some nutrients and ion carriers whose deregulation ensures the necessary reprogramming of energy metabolism in cancer cells. In this study, we investigated the functional role in tumor progression of the sodium/iodide symporter (NIS; aka SLC5A5), which is upregulated and mislocalized in many human carcinomas. Notably, we found that NIS enhanced cell migration and invasion without ion transport being involved. These functions were mediated by NIS binding to leukemia-associated RhoA guanine exchange factor, a Rho guanine exchange factor that activates the small GTPase RhoA. Sequestering NIS in intracellular organelles or impairing its targeting to the cell surface (as observed in many cancers) led to a further increase in cell motility and invasiveness. In sum, our results established NIS as a carrier protein that interacts with a major cell signaling hub to facilitate tumor cell locomotion and invasion.

  4. 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;


    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate....... 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...... 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/PYGM signaling...

  5. Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. (United States)

    Serina, L; Blondin, C; Krin, E; Sismeiro, O; Danchin, A; Sakamoto, H; Gilles, A M; Bârzu, O


    The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF. (United States)

    Perrot, Valerie; Vazquez-Prado, Jose; Gutkind, J Silvio


    Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. Here we show that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, we show that ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration.

  7. The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration. (United States)

    Lessey-Morillon, Elizabeth C; Osborne, Lukas D; Monaghan-Benson, Elizabeth; Guilluy, Christophe; O'Brien, E Timothy; Superfine, Richard; Burridge, Keith


    RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. Although much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared with ICAM-1 clustering alone. We have identified that leukemia-associated Rho guanine nucleotide exchange factor (LARG), also known as Rho GEF 12 (ARHGEF12) acts downstream of clustered ICAM-1 to increase RhoA activity, and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. To our knowledge, this is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes.

  8. Essential role for vav Guanine nucleotide exchange factors in brain-derived neurotrophic factor-induced dendritic spine growth and synapse plasticity. (United States)

    Hale, Carly F; Dietz, Karen C; Varela, Juan A; Wood, Cody B; Zirlin, Benjamin C; Leverich, Leah S; Greene, Robert W; Cowan, Christopher W


    Brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB, regulate a wide range of cellular processes, including dendritic spine formation and functional synapse plasticity. However, the signaling mechanisms that link BDNF-activated TrkB to F-actin remodeling enzymes and dendritic spine morphological plasticity remain poorly understood. We report here that BDNF/TrkB signaling in neurons activates the Vav family of Rac/RhoA guanine nucleotide exchange factors through a novel TrkB-dependent mechanism. We find that Vav is required for BDNF-stimulated Rac-GTP production in cortical and hippocampal neurons. Vav is partially enriched at excitatory synapses in the postnatal hippocampus but does not appear to be required for normal dendritic spine density. Rather, we observe significant reductions in both BDNF-induced, rapid, dendritic spine head growth and in CA3-CA1 theta burst-stimulated long-term potentiation in Vav-deficient mouse hippocampal slices, suggesting that Vav-dependent regulation of dendritic spine morphological plasticity facilitates normal functional synapse plasticity.

  9. Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) Ly-GDI in platelet function: a spatial systems approach. (United States)

    Ngo, Anh T P; Thierheimer, Marisa L D; Babur, Özgün; Rocheleau, Anne D; Huang, Tao; Pang, Jiaqing; Rigg, Rachel A; Mitrugno, Annachiara; Theodorescu, Dan; Burchard, Julja; Nan, Xiaolin; Demir, Emek; McCarty, Owen J T; Aslan, Joseph E


    Upon activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. While RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

  10. Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge. (United States)

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


    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.

  11. Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1. (United States)

    Fleming, Ian N; Batty, Ian H; Prescott, Alan R; Gray, Alex; Kular, Gursant S; Stewart, Hazel; Downes, C Peter


    Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P(2) to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P(2) breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P(3) concentrations, rather than the closely related lipid, PtdIns(3,4)P(2). Finally, the data demonstrate that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.

  12. Role of the guanine nucleotide exchange factor in Akt2-mediated plasma membrane translocation of GLUT4 in insulin-stimulated skeletal muscle. (United States)

    Takenaka, Nobuyuki; Yasuda, Naoto; Nihata, Yuma; Hosooka, Tetsuya; Noguchi, Tetsuya; Aiba, Atsu; Satoh, Takaya


    The small GTPase Rac1 plays a key role in insulin-promoted glucose uptake mediated by the GLUT4 glucose transporter in skeletal muscle. Our recent studies have demonstrated that the serine/threonine protein kinase Akt2 is critically involved in insulin-dependent Rac1 activation. The purpose of this study is to clarify the role of the guanine nucleotide exchange factor FLJ00068 in Akt2-mediated Rac1 activation and GLUT4 translocation in mouse skeletal muscle and cultured myocytes. Constitutively activated FLJ00068 induced GLUT4 translocation in a Rac1-dependent and Akt2-independent manner in L6 myocytes. On the other hand, knockdown of FLJ00068 significantly reduced constitutively activated Akt2-triggered GLUT4 translocation. Furthermore, Rac1 activation and GLUT4 translocation induced by constitutively activated phosphoinositide 3-kinase were inhibited by knockdown of FLJ00068. In mouse gastrocnemius muscle, constitutively activated FLJ00068 actually induced GLUT4 translocation to the sarcolemma. GLUT4 translocation by constitutively activated FLJ00068 was totally abolished in rac1 knockout mouse gastrocnemius muscle. Additionally, we were successful in detecting the activation of Rac1 following the expression of constitutively activated FLJ00068 in gastrocnemius muscle by immunofluorescence microscopy using an activation-specific probe. Collectively, these results strongly support the notion that FLJ00068 regulates Rac1 downstream of Akt2, leading to the stimulation of glucose uptake in skeletal muscle.

  13. Substrate specificity and recognition is conferred by the pleckstrin homology domain of the Dbl family guanine nucleotide exchange factor P-Rex2. (United States)

    Joseph, Raji E; Norris, F A


    Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.

  14. Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)* (United States)

    Carter, Angela M.; Gutowski, Stephen; Sternweis, Paul C.


    The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA. PMID:24855647

  15. Agonist-induced Ca2+ sensitization in smooth muscle: redundancy of Rho guanine nucleotide exchange factors (RhoGEFs) and response kinetics, a caged compound study. (United States)

    Artamonov, Mykhaylo V; Momotani, Ko; Stevenson, Andra; Trentham, David R; Derewenda, Urszula; Derewenda, Zygmunt S; Read, Paul W; Gutkind, J Silvio; Somlyo, Avril V


    Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.

  16. 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.

  17. 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: [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)


    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.

  18. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Takenaka

    Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

  19. 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


    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

  20. Brain regional changes of guanine nucleotide binding protein-inhabitant 2 in acute and chronic morphine-tolerant and-dependent rats

    Institute of Scientific and Technical Information of China (English)

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


    BACKGROUND:Drug addiction involves two main central nervous systems,namely the dopamine and noradrenaline systems.These systems are primarily distributed in five brain regions:the ventrai tegmental area,the nucleus accumbens,the prefrontal coaex,the hippocampus,and the locus coeruleus.OBJECTIVE:To investigate regional changes of guanine nucleotide binding protein-inhabitant 2(Gi2)in dopaminergic and noradrenergic neurons in brains of morphine-tolerant and-dependent rats.DESIGN,TIME,AND SETTING:A randomized centrel study was performed at the Department of Neurobiology in the Second Military Medical University of Chinese PLA(Shanghai,China) between September 2002 and March 2004.MATERIALS:Thirty-six,healthy, male, Sprague-Dawley (SD) rats were used to establish morphine-dependent models.Morphine hydrochloride was a product of Shenyang First Pharmaceutical Factory (China);naloxone hydrochloride was a product of Beijing Four-Ring Pharmaceutical Factory (China);and α subunit of Gt2 antibody was offered by Santa Cruz Biotechnology,Inc(USA).METHODS:Thirty-six SD rats were randomly divided into six groups(n=6):(1)acute morphine-dependent group,(2)acute abstinent group,(3)acute control group,(4)chronic morphine-dependent group,(5)chronic abstinent group,and(6)chronic control group.Rats in the acute morphine-dependent and the acute groups were injected with morphine(5 mg/kg),one injection every two hours,for a total of eight injections.In the acute and chronic morphine-dependent rat models,morphine withdrawal syndrome was precipitated by an injection of naloxone (5 mg/kg).Rats in the acute control group were given a peritoneal iniection of physiological saline at the same administration time as the above two groups.Rats in the chronic morphine-dependent and chronic abstinent groups were injected with morphine three times per day.The administration dose on day 1 was initially 5 mg/kg at 20:00,which increased by 5 mh/kg at 8:00,12:00,and 20:00 until day 7.On day 13,the dose

  1. Inhibition of thyrotropin-stimulated adenosine 3',5'-monophosphate formation in rat thyroid cells by an adenosine analog. Evidence that the inhibition is mediated by the putative inhibitory guanine nucleotide regulatory protein. (United States)

    Berman, M I; Thomas, C G; Nayfeh, S N


    Addition of N6-(L-2-phenylisopropyl)-adenosine (PIA) to cultured FRTL-5 rat thyroid cells led to a concentration-dependent inhibition of TSH-stimulated cAMP formation. Half-maximal inhibition was attained with approximately 0.5 nM PIA. Forskolin and cholera toxin-stimulated cAMP production were also inhibited by PIA. 3-Isobutyl-methylxanthine inhibited the effect of PIA. These results are consistent with the presence of inhibitory adenosine receptors (Ri). Ri-sites were further demonstrated by the binding of 3H-cyclohexyl-adenosine to FRTL-5 plasma membranes. High (Kd = 0.50 +/- 0.07 nM) and low affinity (Kd = 5.95 +/- 2.33 nM) binding sites were observed. Pretreatment of FRTL-5 cells with pertussis, but not cholera, toxin effectively antagonized the inhibitory effects of PIA on cAMP production. ADP-ribosylation of FRTL-5 membranes with [32P]-NAD in the presence of cholera or pertussis toxin specifically labeled a 45,000 and 41,000 Mr species, respectively, which correspond to the alpha subunit of the stimulatory and inhibitory guanine nucleotide regulatory proteins. These results demonstrate that PIA inhibits TSH-stimulated cAMP production via Ri-sites on FRTL-5 thyroid cells. PIA appears to exert its inhibitory effects through the inhibitory guanine nucleotide regulatory protein.

  2. Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm. (United States)

    Wen, Liuying; Fukuda, Masako; Sunada, Mariko; Ishino, Sonoko; Ishino, Yoshizumi; Okita, Thomas W; Ogawa, Masahiro; Ueda, Takashi; Kumamaru, Toshihiro


    Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.

  3. Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers. (United States)

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


    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.

  4. 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;


    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...... suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5-8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4...

  5. Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation – molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor

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    Seki Masaya


    Full Text Available Abstract Background The irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. It is well known that the activation of the small GTPase Rho leads to actin stress fibers assembly. This study was performed to evaluate the effect of simulated microgravity on the activation of Rho that is involved in actin fiber remodeling in cells. Results Clinorotation influences actin fiber remodeling and its related signaling pathways that involve the small GTPase Rho. Actin stress fiber remodeling was significantly inhibited to a greater extent in cells cultured under clinorotation than in static cultured cells. From the gene and protein expression analyses, we found that the expression level of leukemia-associated Rho guanine nucleotide exchange factor (LARG, which activates Rho, was downregulated under clinorotation. Moreover, we identified the full-length LARG cDNA. The amount of GTP-bound RhoA, that is, the active form of RhoA, decreased under this condition. Conclusion The activation of the small GTPase Rho was influenced by simulated microgravity generated by a three-dimensional (3D clinostat. Furthermore, the full-length cDNA of bovine LARG, a member of the Rho guanine nucleotide exchange factor (GEF family, was identified, and its gene expression was observed to be downregulated under clinorotation. This downregulation subsequently resulted in the repression of RhoA activation. These results indicated that the disorganization of the actin fibers was caused by the inhibition of Rho activation by 3D clinorotation.

  6. Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein. (United States)

    Gabig, T G; English, D; Akard, L P; Schell, M J


    Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

  7. The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases. (United States)

    Bergom, Carmen; Hauser, Andrew D; Rymaszewski, Amy; Gonyo, Patrick; Prokop, Jeremy W; Jennings, Benjamin C; Lawton, Alexis J; Frei, Anne; Lorimer, Ellen L; Aguilera-Barrantes, Irene; Mackinnon, Alexander C; Noon, Kathleen; Fierke, Carol A; Williams, Carol L


    The small GTPase DiRas1 has tumor-suppressive activities, unlike the oncogenic properties more common to small GTPases such as K-Ras and RhoA. Although DiRas1 has been found to be a tumor suppressor in gliomas and esophageal squamous cell carcinomas, the mechanisms by which it inhibits malignant phenotypes have not been fully determined. In this study, we demonstrate that DiRas1 binds to SmgGDS, a protein that promotes the activation of several oncogenic GTPases. In silico docking studies predict that DiRas1 binds to SmgGDS in a manner similar to other small GTPases. SmgGDS is a guanine nucleotide exchange factor for RhoA, but we report here that SmgGDS does not mediate GDP/GTP exchange on DiRas1. Intriguingly, DiRas1 acts similarly to a dominant-negative small GTPase, binding to SmgGDS and inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A. DiRas1 is expressed in normal breast tissue, but its expression is decreased in most breast cancers, similar to its family member DiRas3 (ARHI). DiRas1 inhibits RhoA- and SmgGDS-mediated NF-κB transcriptional activity in HEK293T cells. We also report that DiRas1 suppresses basal NF-κB activation in breast cancer and glioblastoma cell lines. Taken together, our data support a model in which DiRas1 expression inhibits malignant features of cancers in part by nonproductively binding to SmgGDS and inhibiting the binding of other small GTPases to SmgGDS.

  8. Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase. (United States)

    Chikumi, Hiroki; Fukuhara, Shigetomo; Gutkind, J Silvio


    A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through

  9. Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G-protein signaling (RGS)-containing Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). (United States)

    Jaiswal, Mamta; Gremer, Lothar; Dvorsky, Radovan; Haeusler, Lars Christian; Cirstea, Ion C; Uhlenbrock, Katharina; Ahmadian, Mohammad Reza


    The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 10(7)-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.

  10. Endogenous melatonin and oxidatively damaged guanine in DNA

    Directory of Open Access Journals (Sweden)

    Poulsen Henrik E


    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

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

    Institute of Scientific and Technical Information of China (English)

    Jun REN; Cindy X FANG


    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.

  12. 鸟嘌呤核苷交换因子Dock180与Elmo1在卵巢癌细胞中的共表达%Co-expression of guanine-nucleotide exchange factor DOCK180 and ELMO1 in ovarian cancer cells

    Institute of Scientific and Technical Information of China (English)

    张瑜; 高一萌; 李文燕; 彭慧娟; 刘斌; 王辉; 令狐华


    目的 寻找卵巢癌细胞中鸟嘌呤核苷交换因子Dock 180与Elmo1呈相互协同作用的依据.方法 采用Westem blot检测人卵巢癌组织、良性肿瘤组织及正常卵巢组织中的Dock 180与Elmo1的表达水平;免疫组化检测卵巢癌组织中Dock 180与Elmol的分布;免疫荧光检测Dock180与Elmo1在卵巢癌细胞SKOV3中的定位分布;检测Dock180表达缺失的SKOV3细胞中内源性Elmo1的表达水平.结果 Dock 180与Elmo1在人卵巢癌组织中的表达水平呈明显正相关(r=0.829,P<0.05),在卵巢癌组织中二者的表达水平显著高于卵巢良性肿瘤组织组和卵巢正常组织组(P<0.05)[Dock180:(1.054±0.114)、(0.518 ±0.126)、(0.425±0.072); Elmo1:(0.864 ±0.114)、(0.374±0.076)、(0.300±0.105)].免疫组化及细胞化学染色均显示二者在人卵巢癌组织和细胞内的分布具有一致性.而且在Dock180表达缺失细胞中,Elmol的表达水平也同时降低.结论 Dock180与Elmo1在促进卵巢癌的癌变过程中可能具有相互协同作用.%Objective To seek evidence to support the synergic effect of guanine-nucleotide exchange factor (GEF) downstream of crk with moleculer weight of 180kDa of human (Dockl80) and engulfment and cell motilityl (Elmol) in ovarian cancer cells. Methods Dockl80 and Elmol expression were detected by Western blotting in ovarian cancer tissues (EOC, n = 29) , benign ovarian tumors (BOT, n = 14) and normal ovary tissues ( Normal, n = 10). Immunohistochemistry was performed to detect the distribution of Dockl80 and Elmol in the ovarian cancer tissues. Immunofluorescence staining was utilized to detect the location and distribution of Dockl80 and Elmol in ovarian cancer cell SKOV3. And finally, endogenous Elmol expression was assayed in Dockl80 knockdown cells, which was established previously. Results Both Dockl80 and Elmol expression presented at higher levels in EOC than those in benign and normal ovarian tissues (P 0. 05). Dockl80 expression

  13. 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;


    , 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...

  14. Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Jérôme Mulhbacher


    Full Text Available Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5'-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1 binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

  15. 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


    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

  16. Nucleotide Metabolism

    DEFF Research Database (Denmark)

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


    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....

  17. Identification of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end product. (United States)

    Papoulis, A; al-Abed, Y; Bucala, R


    Reducing sugars such as glucose react nonenzymatically with protein amino groups to initiate a posttranslational modification process known as advanced glycosylation. Nucleotide bases also participate in advanced glycosylation reactions, producing DNA-linked advanced glycosylation endproducts (AGEs) that cause mutations and DNA transposition. Although several protein-derived AGEs have been isolated and structurally characterized, AGE-modified nucleotides have not yet been reported. We systematically examined the reactivities of the model nucleotide bases 9-methylguanine (9-mG), 9-methyladenine (9-mA), and 1-methylcytosine (1-mC) toward glucose and several glucose-derived reactants. In "fast" reactions performed at refluxing temperature and physiological pH, 1 equiv of nucleotide base was reacted with 10 equiv of D-glucose, D-glucose 6-phosphate (G-6-P), D-glucose 6-phosphate/lysine (G-6-P/Lys), the Schiff base 1-n-propylamino-N-D-glucoside (SB), or the Amadori product 1-n-propylamino-N-D-fructose (AP). In every reaction involving 9-mG, N2-(1-carboxyethyl)-9-methylguanine (CEmG) was a major product which was produced. N2-(1-carboxyethyl)-9-methylguanine also formed from 9-mG and AP in long-term incubations performed at 37 degrees C. Direct treatment of 9-mG with methylglyoxal (MG), a Maillard reaction propagator that forms from the decomposition of AP, also produced CEmG in high yield. N2-(1-Carboxyethyl)-9-methylguanine appears to result from the nucleophilic addition of the primary amino group of guanine to the ketone group of MG followed by an intramolecular rearrangement. Methylglyoxal is a known prokaryotic mutagen and was shown additionally to be mutagenic in a eukaryotic shuttle vector assay system.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. 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


    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).

  19. Nucleotide binding switches the information flow in ras GTPases. (United States)

    Raimondi, Francesco; Portella, Guillem; Orozco, Modesto; Fanelli, Francesca


    The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (S(GDP) and S(GTP), respectively). For all the considered systems, the intrinsic flexibility of S(GDP) was higher than that of S(GTP), suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, S(GDP) of Gα(t), is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP.

  20. Nucleotide binding switches the information flow in ras GTPases.

    Directory of Open Access Journals (Sweden)

    Francesco Raimondi


    Full Text Available The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (S(GDP and S(GTP, respectively. For all the considered systems, the intrinsic flexibility of S(GDP was higher than that of S(GTP, suggesting that Guanine Exchange Factor (GEF recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, S(GDP of Gα(t, is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins, compared to GTP.

  1. Calculation of the Stabilization Energies of Oxidatively Damaged Guanine Base Pairs with Guanine

    Directory of Open Access Journals (Sweden)

    Hiroshi Miyazawa


    Full Text Available DNA is constantly exposed to endogenous and exogenous oxidative stresses. Damaged DNA can cause mutations, which may increase the risk of developing cancer and other diseases. G:C-C:G transversions are caused by various oxidative stresses. 2,2,4-Triamino-5(2H-oxazolone (Oz, guanidinohydantoin (Gh/iminoallantoin (Ia and spiro-imino-dihydantoin (Sp are known products of oxidative guanine damage. These damaged bases can base pair with guanine and cause G:C-C:G transversions. In this study, the stabilization energies of these bases paired with guanine were calculated in vacuo and in water. The calculated stabilization energies of the Ia:G base pairs were similar to that of the native C:G base pair, and both bases pairs have three hydrogen bonds. By contrast, the calculated stabilization energies of Gh:G, which form two hydrogen bonds, were lower than the Ia:G base pairs, suggesting that the stabilization energy depends on the number of hydrogen bonds. In addition, the Sp:G base pairs were less stable than the Ia:G base pairs. Furthermore, calculations showed that the Oz:G base pairs were less stable than the Ia:G, Gh:G and Sp:G base pairs, even though experimental results showed that incorporation of guanine opposite Oz is more efficient than that opposite Gh/Ia and Sp.

  2. 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)


    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.

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

    Directory of Open Access Journals (Sweden)

    Cristian eDroppelmann


    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.

  4. 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


    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.

  5. Endogenous melatonin and oxidatively damaged guanine in DNA

    DEFF Research Database (Denmark)

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


    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...... of oxidatively damaged guanine in DNA, thereby possibly increasing the risk of developing cancer. The possible different effects of melatonin in the rates of utilization of pathways for repair of oxidatively damaged guanine in DNA identified between older women and older men are intriguing....

  6. Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

    NARCIS (Netherlands)

    Van Leeuwen, Frank N; Olivo, Cristina; Grivell, Shula; Giepmans, Ben N G; Collard, John G; Moolenaar, Wouter H


    Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas R

  7. Guanine Nucleotides Modulate Cell Surface cAMP-Binding Sites in Membranes from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van


    D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, t½ = 15 s). A second class is fast dissociating (t½ about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low a

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

    Directory of Open Access Journals (Sweden)

    Zhilong Wang


    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

  9. 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.

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

    Indian Academy of Sciences (India)

    Bhabesh Chandra Deka; Pradip Kr Bhattacharyya


    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.

  11. 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.


    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.

  12. Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase (United States)

    Terán, David; Hocková, Dana; Česnek, Michal; Zíková, Alena; Naesens, Lieve; Keough, Dianne T.; Guddat, Luke W.


    Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed. PMID:27786284

  13. Calculating Distortions of Short DNA Duplexes with Base Pairing Between an Oxidatively Damaged Guanine and a Guanine

    Directory of Open Access Journals (Sweden)

    Masayo Suzuki


    Full Text Available DNA is constantly being oxidized, and oxidized DNA is prone to mutation; moreover, guanine is highly sensitive to several oxidative stressors. Several oxidatively damaged forms of guanine—including 2,2,4-triamino-5(2H-oxazolone (Oz, iminoallantoin (Ia, and spiroiminodihydantoin (Sp—can be paired with guanine, and cause G:C-C:G transversions. Previous findings indicate that guanine is incorporated more efficiently opposite Oz than opposite Ia or Sp, and that these differences in efficiency cannot be explained by differences in the stabilities of G:Oz, G:Ia, and G:Sp base pairs calculated ab initio. Here, to explain previous experimental result, we used a 3-base-pair model DNA duplex to calculate the difference in the stability and the distortion of DNA containing a G:Oz, G:Ia, or G:Sp base pair. We found that the stability of the structure containing 5ꞌ and 3ꞌ base pairs adjacent to G:Oz was more stable than that containing the respective base pairs adjacent to G:Ia or G:Sp. Moreover, the distortion of the structure in the DNA model duplex that contained a G:Oz was smaller than that containing a G:Ia or G:Sp. Therefore, our discussion can explain the previous results involving translesion synthesis past an oxidatively damaged guanine.

  14. Complex conformational heterogeneity of the highly flexible O6-benzyl-guanine DNA adduct. (United States)

    Wilson, Katie A; Wetmore, Stacey D


    The conformational preference of the O6-benzyl-guanine (BzG) adduct was computationally examined using nucleoside, nucleotide, and DNA models, which provided critical information about the potential mutagenic consequences and toxicity of the BzG adduct in our cells. Substantial conformational flexibility of the BzG moiety, including rotation of the bulky group with respect to the base and the internal conformation of the bulk moiety, is seen in the nucleoside and nucleotide models. This large conformational flexibility suggests the conformation adopted by BzG is dependent on the local environment of the BzG adduct. Upon incorporation of the adduct into the DNA helix, the BzG conformational flexibility is maintained. The range of BzG conformations adopted in DNA likely arises due to a combination of the long and flexible (-CH2-) linker, the small adduct size, and the lack of discrete interactions between the bulky moiety and G. Because of the conformational flexibility of the adduct, many DNA conformations are observed for BzG adducted DNA, including those not previously reported in the literature, and thus, a modified nomenclature for adducted DNA conformations is presented. Furthermore, the preferred conformation of BzG adducted DNA is greatly dependent on a number of factors, including the pairing nucleotide, the discrete interactions in the helix, and the solvation of the benzyl moiety. These factors in turn lead to a complicated mutagenic and toxic profile that may invoke pairing with natural C, mispairs, or deletion mutations, which is supported by previously reported experimental biochemical studies. Despite this complex mutagenic profile, pairing with C leads to the most stable helical structure, which is the first combined structural and energetic explanation for experimental studies reporting a higher rate of C incorporation than any other nucleobase upon BzG replication.

  15. High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay. (United States)

    Evelyn, Chris R; Ferng, Timothy; Rojas, Rafael J; Larsen, Martha J; Sondek, John; Neubig, Richard R


    Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.

  16. Classifying Coding DNA with Nucleotide Statistics

    Directory of Open Access Journals (Sweden)

    Nicolas Carels


    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.

  17. Effect of base pairing on the electrochemical oxidation of guanine. (United States)

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


    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.

  18. B3LYP, BLYP and PBE DFT band structures of the nucleotide base stacks (United States)

    Szekeres, Zs; Bogár, F.; Ladik, J.

    DFT crystal orbital (band structure) calculations have been performed for the nucleotide base stacks of cytosine, thymine, adenine, and guanine arranged in DNA B geometry. The band structures obtained with PBE, BLYP, and B3LYP functionals are presented and compared to other related experimental and theoretical results. The influence of the quality of the basis set on the fundamental gap values was also investigated using Clementi's double ζ, 6-31G and 6-31G* basis sets.

  19. In vivo formation of N7-guanine DNA adduct by safrole 2',3'-oxide in mice. (United States)

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


    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.

  20. Crystal Structure of a Replicative DNA Polymerase Bound to the Oxidized Guanine Lesion Guanidinohydantoin

    Energy Technology Data Exchange (ETDEWEB)

    Aller, Pierre; Ye, Yu; Wallace, Susan S.; Burrows, Cynthia J.; Doubli, Sylvie (Vermont); (Utah)


    The oxidation of guanine generates one of the most common DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxoG). The further oxidation of 8-oxoG can produce either guanidinohydantoin (Gh) in duplex DNA or spiroiminodihydantoin (Sp) in nucleosides and ssDNA. Although Gh can be a strong block for replicative DNA polymerases such as RB69 DNA polymerase, this lesion is also mutagenic: DNA polymerases bypass Gh by preferentially incorporating a purine with a slight preference for adenine, which results in G {center_dot} C {yields} T {center_dot} A or G {center_dot} C {yields} C {center_dot} G transversions. The 2.15 {angstrom} crystal structure of the replicative RB69 DNA polymerase in complex with DNA containing Gh reveals that Gh is extrahelical and rotated toward the major groove. In this conformation Gh is no longer in position to serve as a templating base for the incorporation of an incoming nucleotide. This work also constitutes the first crystallographic structure of Gh, which is stabilized in the R configuration in the two polymerase/DNA complexes present in the crystal asymmetric unit. In contrast to 8-oxoG, Gh is found in a high syn conformation in the DNA duplex and therefore presents the same hydrogen bond donor and acceptor pattern as thymine, which explains the propensity of DNA polymerases to incorporate a purine opposite Gh when bypass occurs.

  1. The experimental and theoretical gas phase acidities of adenine, guanine, cytosine, uracil, thymine and halouracils (United States)

    Chen, Edward C. M.; Herder, Charles; Chen, Edward S.


    The gas phase acidities GPA (Δ H (298) for deprotonation) of the most stable tautomers of adenine, guanine, cytosine, uracil and thymine are evaluated. New GPA are obtained from electron impact spectra and acid dissociation constants measured in dimethylsulfoxide for A, U and 5-FU. The average experimental GPA are: [N1 sbnd H] C 340(2); T 333(2); U 333(2); 5-FU 329(4); [N9 sbnd H] A 333(1); G 332(4); all in kcal/mol. Only cytosine is a weaker acid than HCl in the gas phase. The most acidic hydrogens in the nucleotides are replaced by the sugar in DNA and RNA. The experimental N3 sbnd H GPA are G 334(4); U 347(2), T 347(4), while the predicted N3 sbnd H 5-FU GPA is 343 kcal/mol. The NH sbnd H GPA are: C 346(4); A 352(2); G 336(4) (all in kcal/mol). These are supported by semi-empirical multiconfiguration configuration interaction calculations. The predicted C8 sbnd H acidities of G and A and the C6 sbnd H of T are about the same, 360(2) kcal/mol. The remaining CH acidities are 370-380 kcal/mol. The 5-halouracils are predicted to be more acidic than HCl.

  2. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide (United States)

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


    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.

  3. Main: Nucleotide Analysis [KOME

    Lifescience Database Archive (English)

    Full Text Available Nucleotide Analysis Japonica genome blast search result Result of blastn search against japon...ica genome sequence kome_japonica_genome_blast_search_result ...

  4. Characterization of nucleotide misincorporation patterns in the iceman's mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Cristina Olivieri

    Full Text Available BACKGROUND: The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation ("miscoding lesions" has been the object of extensive investigations. METHODOLOGY/PRINCIPAL FINDINGS: To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp mitochondrial (mt DNA sequences obtained from the 5,350-5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Otzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85% of the observed nucleotide misincorporations in ancient sequences. CONCLUSIONS/SIGNIFICANCE: This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine-->thymine/guanine

  5. Ubiquinol (QH(2)) functions as a negative regulator of purine nucleotide inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein. (United States)

    Woyda-Ploszczyca, Andrzej; Jarmuszkiewicz, Wieslawa


    We compared the influence of different adenine and guanine nucleotides on the free fatty acid-induced uncoupling protein (UCP) activity in non-phosphorylating Acanthamoeba castellanii mitochondria when the membranous ubiquinone (Q) redox state was varied. The purine nucleotides exhibit an inhibitory effect in the following descending order: GTP>ATP>GDP>ADP≫GMP>AMP. The efficiency of guanine and adenine nucleotides to inhibit UCP-sustained uncoupling in A. castellanii mitochondria depends on the Q redox state. Inhibition by purine nucleotides can be increased with decreasing Q reduction level (thereby ubiquinol, QH₂ concentration) even with nucleoside monophosphates that are very weak inhibitors at the initial respiration. On the other hand, the inhibition can be alleviated with increasing Q reduction level (thereby QH₂ concentration). The most important finding was that ubiquinol (QH₂) but not oxidised Q functions as a negative regulator of UCP inhibition by purine nucleotides. For a given concentration of QH₂, the linoleic acid-induced GTP-inhibited H(+) leak was the same for two types of A. castellanii mitochondria that differ in the endogenous Q content. When availability of the inhibitor (GTP) or the negative inhibition modulator (QH₂) was changed, a competitive influence on the UCP activity was observed. QH₂ decreases the affinity of UCP for GTP and, vice versa, GTP decreases the affinity of UCP for QH₂. These results describe the kinetic mechanism of regulation of UCP affinity for purine nucleotides by endogenous QH₂ in the mitochondria of a unicellular eukaryote.

  6. Analysis of difference spectra of protonated DNA: determination of degree of protonation of nitrogen bases and the fractions of disordered nucleotide pairs. (United States)

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


    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Caroline Kisker


    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.

  8. Single Nucleotide Polymorphism

    DEFF Research Database (Denmark)

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


    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 and bri...

  9. Statistical analysis of nucleotide runs in coding and noncoding DNA sequences. (United States)

    Sprizhitsky YuA; Nechipurenko YuD; Alexandrov, A A; Volkenstein, M V


    A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.

  10. Cytochrome b nucleotide sequence variation among the Atlantic Alcidae. (United States)

    Friesen, V L; Montevecchi, W A; Davidson, W S


    Analysis of cytochrome b nucleotide sequences of the six extant species of Atlantic alcids and a gull revealed an excess of adenines and cytosines and a deficit of guanines at silent sites on the coding strand. Phylogenetic analyses grouped the sequences of the common (Uria aalge) and Brünnich's (U. lomvia) guillemots, followed by the razorbill (Alca torda) and little auk (Alle alle). The black guillemot (Cepphus grylle) sequence formed a sister taxon, and the puffin (Fratercula arctica) fell outside the other alcids. Phylogenetic comparisons of substitutions indicated that mutabilities of bases did not differ, but that C was much more likely to be incorporated than was G. Imbalances in base composition appear to result from a strand bias in replication errors, which may result from selection on secondary RNA structure and/or the energetics of codon-anticodon interactions.

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

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


    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.

  12. A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana (United States)

    Rop/Rac small GTPases are central to diverse developmental and cellular activities in plants, playing an especially important Role in polar growth of pollen tubes. Although it is established that a class of plant-specific RopGEFs promotes the activity of Rop/Rac through the catalytic PRONE (Plant-sp...

  13. Activated RhoA is a positive feedback regulator of the Lbc family of Rho guanine nucleotide exchange factor proteins. (United States)

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


    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.

  14. Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins* (United States)

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


    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. PMID:23493395

  15. G-quartet type self-assembly of guanine functionalized single-walled carbon nanotubes (United States)

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


    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

  16. Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

    Directory of Open Access Journals (Sweden)

    Oliva Glaucius


    Full Text Available Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT (EC 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.

  17. Theoretical Study of Hydrated Cd~(2+) Interactions with Guanine

    Institute of Scientific and Technical Information of China (English)

    王敏; 洒荣建; 吴克琛; 李巧红; 韦永勤


    Theoretical study was performed to investigate how the hydration of cadmium ca-tion influences the structure and properties of guanine.The aqueous environment was simulated by both explicit solvent(1-5 water molecules) model and implicit solvent model.For complexes in which Cd2+ attached to the N(7) and O(6) sites of guanine,energy analysis together with the Natural Bonding Orbital(NBO) analysis were performed to elucidate the bonding characteristics in detail.The most stable structures are penta-coordinate complexes without aqua ligand located at the guanine site.Higher number of water ligands corresponds to higher stabilization energies.Average bonding energies of G-Cd increase with the number of water molecules.Bonding energies of water ligands depend on its position in the complexes.The charge distribution of guanine changed with increasing the number of water ligands,which may also influence the base-pairing pattern of guanine.There is positive charge transfer from guanine to aqua ligand as the number of the hydration waters increases.IEFPCM optimization has results comparable to the [CdG(H2O)5]2+ structure 5a.

  18. 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


    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.

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


    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. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes. (United States)

    Shi, Xianzong; Jarvis, Donald L


    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.

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


    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.

  2. Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand. (United States)

    Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao


    A novel adenine (Ad) fluorescence probe (Eu(III)-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the Eu(III)-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the Eu(III)-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the Eu(III)-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using Eu(III)-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of Eu(III)-dtpa-bis(guanine) at 570nm as a function of adenine (Ad) concentration in the range of 0.00-5.00×10(-5)molL(-1) was observed. The detection limit is about 4.70×10(-7)molL(-1).

  3. Electrochemical studies on the oxidation of guanine and adenine at cyclodextrin modified electrodes. (United States)

    Abbaspour, Abdolkarim; Noori, Abolhassan


    An electrochemical sensor for guanine and adenine using cyclodextrin-modified poly(N-acetylaniline) (PNAANI) on a carbon paste electrode has been developed. The oxidation mechanism of guanine and adenine on the surface of the electrode was investigated by cyclic voltammetry. It was found that the electrode processes are irreversible, pH dependent, and involve several reaction products. The electron transfer process occurs in consecutive steps with the formation of a strongly adsorbed intermediate on the electrode surface. Also, a new method for estimating the apparent formation constants of guanine and adenine with the immobilized cyclodextrins, through the change of surface coverage of studied analytes has been reported. Both guanine and adenine showed linear concentrations in the range of 0.1-10 microM by using differential pulse voltammetry, with an experimental limit of detection down to 0.05 microM. Linear concentration ranges of 2-150 microM for guanine and 6-104 microM for adenine have been found when cyclic voltammetry was used for determination of both analytes.

  4. Guanine-based structural coloration as an indicator of oxidative stress in a cichlid fish. (United States)

    Cahn, Matthew D; Brown, Alexandria C; Clotfelter, Ethan D


    Vertebrate pigmentation is known to be influenced by oxidative stress, but few studies have tested the hypothesis that structural coloration can be similarly affected. We tested whether fish iridophores, which produce structural color using guanine stacks, might be affected by the prooxidant-antioxidant balance of the animal. Specifically, we hypothesized that convict cichlids (Amatitlania nigrofasciata) metabolize guanine present in iridophores to uric acid, an antioxidant, in response to oxidative damage. We used Hunter's contrast gloss and high performance liquid chromatography to determine whether dietary guanine supplementation allows fish to maintain their structural coloration despite oxidative stress induced via ultraviolet-B (UV-B) radiation. We found that dietary guanine was associated with greater skin gloss, and that exposure to UV-B light reduced glossiness. UV-B exposure did not increase oxidative damage (acrolein) or total antioxidant capacity in the skin or liver. Our experiment did not detect effects of dietary guanine or UV-B light on uric acid, but uric acid was positively related to antioxidant capacity. Our results support the hypothesis that structural color in fish may be altered by environmental stressors such as exposure to UV light, and highlight the need for future studies to consider the role of iridophores in condition-dependent visual signaling.

  5. Theoretical Study of the Photophysics of 8-Vinylguanine, an Isomorphic Fluorescent Analogue of Guanine. (United States)

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


    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.

  6. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara


    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.

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

    Directory of Open Access Journals (Sweden)

    Puig Juan G


    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

  8. Synthesis and nonenzymatic template-directed polymerization of 2'-amino-2'-deoxythreose nucleotides. (United States)

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


    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.

  9. High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Jan Duchek


    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.

  11. Mechanisms of oxidation of guanine in DNA by carbonate radical anion, a decomposition product of nitrosoperoxycarbonate. (United States)

    Lee, Young Ae; Yun, Byeong Hwa; Kim, Seog K; Margolin, Yelena; Dedon, Peter C; Geacintov, Nicholas E; Shafirovich, Vladimir


    Peroxynitrite is produced during inflammation and combines rapidly with carbon dioxide to yield the unstable nitrosoperoxycarbonate, which decomposes (in part) to CO(3) (.-) and (.)NO(2) radicals. The CO(3) (.-) radicals oxidize guanine bases in DNA through a one-electron transfer reaction process that ultimately results in the formation of stable guanine oxidation products. Here we have explored these mechanisms, starting with a spectroscopic study of the kinetics of electron transfer from 20-22mer double-stranded oligonucleotides to CO(3) (.-) radicals, together with the effects of base sequence on the formation of the end-products in runs of one, two, or three contiguous guanines. The distributions of these alkali-labile lesions were determined by gel electrophoresis methods. The cascade of events was initiated through the use of 308 nm XeCl excimer laser pulses to generate CO(3) (.-) radicals by an established method based on the photodissociation of persulfate to sulfate radicals and the oxidation of bicarbonate. Although the Saito model (Saito et al., J. Am. Chem. Soc. 1995, 117, 6406-6407) predicts relative ease of one-electron oxidations in DNA, following the trend 5'-GGG > 5'-GG > 5'-G, we found that the rate constants for CO(3) (.-)-mediated oxidation of guanines in these sequence contexts (k(5)) showed only small variation within a narrow range [(1.5-3.0)x10(7) M(-1) s(-1)]. In contrast, the distributions of the end-products are dependent on the base sequence context and are higher at the 5'-G in 5'-GG sequences and at the first two 5'-guanines in the 5'-GGG sequences. These effects are attributed to a combination of initial hole distributions among the contiguous guanines and the subsequent differences in chemical reaction yields at each guanine. The lack of dependence of k(5) on sequence context indicates that the one-electron oxidation of guanine in DNA by CO(3) (.-) radicals occurs by an inner-sphere mechanism.

  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.


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

    Directory of Open Access Journals (Sweden)

    J. Zheng


    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.

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

    NARCIS (Netherlands)

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


    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

  15. Nucleotide binding affects intrinsic dynamics and structural communication in Ras GTPases. (United States)

    Fanelli, Francesca; Raimondi, Francesco


    The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. These proteins act biologically as molecular switches, which, cycling between OFF and ON states, play fundamental role in cell biology. This review article summarizes the inferences from the widest computational analyses done so far on Ras GTPases aimed at providing a comprehensive structural/dynamic view of the trans-family and family-specific functioning mechanisms. These variegated comparative analyses could infer the evolutionary and intrinsic flexibilities as well as the structural communication features in the most representative G protein families in different functional states. In spite of the low sequence similarities, the members of the Ras superfamily share the topology of the Ras-like domain, including the nucleotide binding site. GDP and GTP make very similar interactions in all GTPases and differences in their binding modes are localized around the γ-phosphate of GTP. Remarkably, such subtle local differences result in significant differences in the functional dynamics and structural communication features of the protein. In Ras GTPases, the nucleotide plays a central and active role in dictating functional dynamics, establishing the major structure network, and mediating the communication paths instrumental in function retention and specialization. Collectively, the results of these studies support the speculation that an "extended conformational selection model" that embraces a repertoire of selection and adjustment processes is likely more suitable to describe the nucleotide behavior in these important molecular switches.

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

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


    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)].

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

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


    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.

  18. Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study. (United States)

    Jain, Swapan S; Sonavane, Uddhavesh B; Uppuladinne, Mallikarjunachari V N; McLaughlin, Emily C; Wang, Weiqing; Black, Sheneil; Joshi, Rajendra R


    Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.

  19. The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta. (United States)

    Choi, Jung-Suk; Dasari, Anvesh; Hu, Peter; Benkovic, Stephen J; Berdis, Anthony J


    This report evaluates the pro-mutagenic behavior of 8-oxo-guanine (8-oxo-G) by quantifying the ability of high-fidelity and specialized DNA polymerases to incorporate natural and modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases such as pol δ and the bacteriophage T4 DNA polymerase replicating 8-oxo-G in an error-prone manner, they display remarkably low efficiencies for TLS compared to normal DNA synthesis. In contrast, pol η shows a combination of high efficiency and low fidelity when replicating 8-oxo-G. These combined properties are consistent with a pro-mutagenic role for pol η when replicating this DNA lesion. Studies using modified nucleotide analogs show that pol η relies heavily on hydrogen-bonding interactions during translesion DNA synthesis. However, nucleobase modifications such as alkylation to the N2 position of guanine significantly increase error-prone synthesis catalyzed by pol η when replicating 8-oxo-G. Molecular modeling studies demonstrate the existence of a hydrophobic pocket in pol η that participates in the increased utilization of certain hydrophobic nucleotides. A model is proposed for enhanced pro-mutagenic replication catalyzed by pol η that couples efficient incorporation of damaged nucleotides opposite oxidized DNA lesions created by reactive oxygen species. The biological implications of this model toward increasing mutagenic events in lung cancer are discussed.

  20. Pt(Ⅱ), Pd(Ⅱ) and Ni(Ⅱ) Complexes Binding to the N(7) Position of Guanine: Influence on the Guanine and Watson-crick GC Base Pair

    Institute of Scientific and Technical Information of China (English)

    章志强; 周立新; 和芹; 赵亚英


    Comprehensive ab initio calculations were performed on the coordination of Pt(II), Pd(II) and Ni(II) adducts to the N(7) of guanine and guanine-cytosine (GC) base pair at the DFT level. The fully optimized geometries of the metal complexes were obtained and the stabilization energies of the interaction between metal adducts and nucleobase were calculated with B3LYP method by using 6-31* basis set for the light atom. While the effective core potential (ECP) is used for metal cation. The results show that both cispalladium and cisnickel cause similar geometric changes of the base pair as cisplatin. For the coordination of metal adducts to guanine, platinum adduct possesses the highest stabilization energy; but the interaction between metal-guanine and cytosine for nickel is larger than that for platinum and palladium. It is worthy to note that hydrolysis effect can also cause significant changes in H-bonds.

  1. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van


    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

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

    Institute of Scientific and Technical Information of China (English)


    @@ Introduction Hypoxanthine-guanine phosphoribosyltransferase ( HGPRT, EC ) 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].

  3. N7-(carboxymethyl)guanine-Lithium Crystalline Complex: A Bioinspired Solid Electrolyte (United States)

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


    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.

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

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


    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.

  5. European Nucleotide Archive in 2016 (United States)

    Toribio, Ana Luisa; Alako, Blaise; Amid, Clara; Cerdeño-Tarrága, Ana; Clarke, Laura; Cleland, Iain; Fairley, Susan; Gibson, Richard; Goodgame, Neil; ten Hoopen, Petra; Jayathilaka, Suran; Kay, Simon; Leinonen, Rasko; Liu, Xin; Martínez-Villacorta, Josué; Pakseresht, Nima; Rajan, Jeena; Reddy, Kethi; Rosello, Marc; Silvester, Nicole; Smirnov, Dmitriy; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy


    The European Nucleotide Archive (ENA; offers a rich platform for data sharing, publishing and archiving and a globally comprehensive data set for onward use by the scientific community. With a broad scope spanning raw sequencing reads, genome assemblies and functional annotation, the resource provides extensive data submission, search and download facilities across web and programmatic interfaces. Here, we outline ENA content and major access modalities, highlight major developments in 2016 and outline a number of examples of data reuse from ENA. PMID:27899630

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

    Energy Technology Data Exchange (ETDEWEB)

    Minaev, B. F., E-mail:, E-mail: [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)


    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.

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

    Indian Academy of Sciences (India)

    Swathi Kota; Hari S Misra


    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.

  8. Ab Initio Study of the Electron Transfer in an Ionized Stacked Complex of Guanines (United States)

    Cauët, Emilie; Liévin, Jacques


    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.

  9. CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes for electrochemical determination of guanine and adenine

    Energy Technology Data Exchange (ETDEWEB)

    Wei Yan [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Huang Qinan [Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Li Maoguo [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Huang Xingjiu [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei 230031 (China); Fang Bin, E-mail: [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Wang Lun, E-mail: [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China)


    Sub-10 nm CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes has been constructed for electrochemial determination of guanine and adenine. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to characterize the nanoparticles CeO{sub 2}/MWCNTs. Electrochemical impedance spectroscopy (EIS) was used to characterize the electrode modifying process. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to study the electrocatalytic activity toward the electrochemical oxidation of guanine and adenine. The detection limit (S/N = 3) for adenine and guanine was found to be 20 and 10 nM, respectively. The obtained sensitivity toward guanine and adenine was 1.26 and 1.13 {mu}A/{mu}M in the linear concentration range 5-50 {mu}M and 5-35 {mu}M, respectively. These results demonstrate that the carbon nanotubes could provide huge locations and facilitate the adsorptive accumulation of the guanine and adenine, and the CeO{sub 2} nanoparticles are promising substrates for the development of high-performance electrocatalysts for biosensing.

  10. Simultaneous Determination of Adenine and Guanine Using Cadmium Selenide Quantum Dots-Graphene Oxide Nanocomposite Modified Electrode. (United States)

    Kalaivani, Arumugam; Narayanan, Sangilimuthu Sriman


    A novel electrochemical sensor was fabricated by immobilizing Cadmium Selenide Quantum Dots (CdSe QDs)-Graphene Oxide (GO) nanocomposite on a paraffin wax impregnated graphite electrode (PIGE) and was used for the simultaneous determination of adenine and guanine. The CdSe QDs-GO nanocomposite was prepared by ultrasonication and was characterized with spectroscopic and microscopic techniques. The nanocomposite modified electrode was characterized by cyclic voltammetry (CV). The modified electrode showed excellent electrocatalytic activity towards the oxidative determination of adenine and guanine with a good peak separation of 0.31 V. This may be due to the high surface area and fast electron transfer kinetics of the nanocomposite. The modified electrode exhibited wide linear ranges from 0.167 μM to 245 μM for Guanine and 0.083 μM to 291 μM for Adenine with detection limits of 0.055 μM Guanine and 0.028 μM of Adenine (S/N = 3) respectively. Further, the modified electrode was used for the quantitative determination of adenine and guanine in herring sperm DNA with satisfactory results. The modified electrode showed acceptable selectivity, reproducibility and stability under optimal conditions.

  11. Effect of hydration on the lowest singlet PiPi* excited-state geometry of guanine: a theoretical study. (United States)

    Shukla, M K; Leszczynski, Jerzy


    An ab-initio computational study was performed to investigate the effect of explicit hydration on the ground and lowest singlet PiPi* excited-state geometry and on the selected stretching vibrational frequencies corresponding to the different NH sites of the guanine acting as hydrogen-bond donors. The studied systems consisted of guanine interacting with one, three, five, six, and seven water molecules. Ground-state geometries were optimized at the HF level, while excited-state geometries were optimized at the CIS level. The 6-311G(d,p) basis set was used in all calculations. The nature of potential energy surfaces was ascertained via the harmonic vibrational frequency analysis; all structures were found minima at the respective potential energy surfaces. The changes in the geometry and the stretching vibrational frequencies of hydrogen-bond-donating sites of the guanine in the ground and excited state consequent to the hydration are discussed. It was found that the first solvation shell of the guanine can accommodate up to six water molecules. The addition of the another water molecule distorts the hydrogen-bonding network by displacing other neighboring water molecules away from the guanine plane.

  12. Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog. (United States)

    Ster, C; Allard, M; Boulanger, S; Lamontagne Boulet, M; Mulhbacher, J; Lafontaine, D A; Marsault, E; Lacasse, P; Malouin, F


    Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following

  13. Nucleotide Metabolism and DNA Replication. (United States)

    Warner, Digby F; Evans, Joanna C; Mizrahi, Valerie


    The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.

  14. Oxidation of Guanine by Carbonate Radicals Derived from Photolysis of Carbonatotetramminecobalt(III) Complexes and the pH Dependence of Intrastrand DNA Cross-links Mediated by Guanine Radical Reactions


    Crean, Conor; Lee, Young Ae; Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir


    The carbonate radical anion CO3•− is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO3•− radicals is known to yield spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), and a novel intrastrand cross-linked product, 5’-d(CCATCG*CT*ACC) between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et ...

  15. Electrocatalytic activity of oxidation products of guanine and 5'-GMP towards the oxidation of NADH

    Energy Technology Data Exchange (ETDEWEB)

    Santos-Alvarez, Noemi de los; Lobo-Castanon, Maria Jesus; Miranda-Ordieres, Arturo J. [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, Julian Claveria 8, 33006 Oviedo (Spain); Tunon-Blanco, Paulino [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)], E-mail:


    We have studied the potential electrocatalytic activity towards the oxidation of NADH of several oxidation products of guanine and its derivative guanosine-5'-monophosphate (5'-GMP) on pyrolytic graphite electrodes (PGE). The distribution of products generated strongly depends on the experimental conditions. Our investigations focused on the oxidation products that are adsorbed on the electrode surface, are redox active and, exhibited electrocatalytic activity toward the oxidation of NADH. These compounds were electrochemically and kinetically characterized in terms of dependence of the formal potential on pH and electron transfer rate constant (k{sub s}). The voltammetric and catalytic behavior of both guanine and 5'-GMP oxidation products was compared with that of other guanine derivatives we have previously studied. Some mechanistic aspects concerning the generation of the catalysts are also discussed.

  16. N7-guanine adducts of the epoxy metabolites of 1,3-butadiene in mice lung. (United States)

    Koivisto, P; Peltonen, K


    Epoxy metabolites of 1,3-butadiene are electrophilic and can bind to nucleophilic sites in DNA forming DNA adducts. In this study, guanine N7 adducts of epoxy butene and guanine N7 adducts of epoxy butanediol were measured in lung tissues of mice inhalation exposed to various concentrations of 1,3-butadiene. 32P-postlabeling of DNA adducts were used to demonstrate that the DNA adducts derived from epoxybutene and epoxybutanediol were formed in a dose dependent manner. More than 98% of all adducts detected were formed from epoxybutanediol. Enantiomeric distribution of the adducts formed in vivo differs from that of in vitro experiments demonstrated before. In the case of epoxybutene most of the adducts were formed to the terminal carbon of the S-epoxybutene enantiomer. Most of the adducts derived from epoxybutanediol were formed from the 2S-3R enantiomer. The data demonstrates that enzymatic processes involved with activation and/or detoxification of the metabolites are enantiospecific and/or DNA repair machinery repairs the damage with stereochemical considerations. These are the crucial factors if interspecies differences in tumor sensitiveness is concerned.

  17. New investigations of the guanine trichloro cuprate(II) complex crystal (United States)

    Fabijanić, Ivana; Matković-Čalogović, Dubravka; Pilepić, Viktor; Ivanišević, Irena; Mohaček-Grošev, Vlasta; Sanković, Krešimir


    Crystals of the guanine trichloro cuprate(II) complex, (HGua)2[Cu2Cl6]·2H2O (HGua = protonated guanine), were prepared and analysed by spectroscopic (IR, Raman) and computational methods. A new single-crystal X-ray diffraction analysis was conducted to obtain data with lower standard uncertainties than those in the previously published structure. Raman and IR spectroscopy and quantum-mechanical analysis gave us new insight into the vibrational states of the (HGua)2[Cu2Cl6]·2H2O crystal. The vibrational spectra of the crystal were assigned by performing a normal coordinate analysis for a free dimer with a centre of inversion as the only symmetry element. The stretching vibration observed at 279 cm-1 in the infrared spectrum corresponds to the N-Cu bond. The noncovalent interaction (NCI) plots and quantum theory of atoms in molecules (QTAIM) analysis of the electron density obtained from periodic DFT calculations elucidated the interactions that exist within the crystal structure. Closed-shell ionic attractions, as well as weak and medium strength hydrogen bonds, prevailed in the crystal packing.

  18. Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation. (United States)

    Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferrucio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S


    Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

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


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


    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 ...

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

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


    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.

  1. Activation of a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein during desensitization of Dictyostelium discoideum cells to chemotactic signals

    NARCIS (Netherlands)

    Snaar-Jagalska, B. Ewa; Es, Saskia van; Kesbeke, Fanja; Haastert, Peter J.M. van


    The chemoattractant cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum. Upon prolonged incubation with cAMP, cells become desensitized via two distinct processes: a decrease in the number of available cAMP-binding sites (down regulation) and modification of the receptor (pr

  2. Polymerase recognition of 2-thio-iso-guanine·5-methyl-4-pyrimidinone (iGs·P)--A new DD/AA base pair. (United States)

    Lee, Dong-Kye; Switzer, Christopher


    Polymerase specificity is reported for a previously unknown base pair with a non-standard DD/AA hydrogen bonding pattern: 2-thio-iso-guanine·5-methyl-4-pyrimidinone. Our findings suggest that atomic substitution may provide a solution for low fidelity previously associated with enzymatic copying of iso-guanine.

  3. Studies by Near Edge X-ray Absorption Spectroscopies of Bonding Dynamics at the Graphene/Guanine Interface - A Proposal for High Mobility, Organic Graphene Field Effect Transistors (United States)


    AFRL-AFOSR-UK-TR-2015-0034 Studies by Near Edge X-ray Absorption Spectroscopies of Bonding Dynamics at the Graphene/Guanine...April 2015 4. TITLE AND SUBTITLE Studies by Near Edge X-ray Absorption Spectroscopies of Bonding Dynamics at the Graphene/Guanine Interface - A

  4. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions (United States)

    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.


    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  5. Molecular basis for nucleotide-binding specificity: role of the exocyclic amino group "N2" in recognition by a guanylyl-ribonuclease. (United States)

    Schrift, Greta L; Waldron, Travis T; Timmons, Mitchell A; Ramaswamy, S; Kearney, William R; Murphy, Kenneth P


    Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (>3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP

  6. Pathways of arachidonic acid peroxyl radical reactions and product formation with guanine radicals. (United States)

    Crean, Conor; Geacintov, Nicholas E; Shafirovich, Vladimir


    Peroxyl radicals were derived from the one-electron oxidation of polyunsaturated fatty acids by sulfate radicals that were generated by the photodissociation of peroxodisulfate anions in air-equilibrated aqueous solutions. Reactions of these peroxyl and neutral guanine radicals, also generated by oxidation with sulfate radicals, were investigated by laser kinetic spectroscopy, and the guanine oxidation products were identified by HPLC and mass spectrometry methods. Sulfate radicals rapidly oxidize arachidonic (ArAc), linoleic (LnAc), and palmitoleic (PmAc) acids with similar rate constants, (2-4) x 10 (9) M (-1) s (-1). The C-centered radicals derived from the oxidation of ArAc and LnAc include nonconjugated Rn(.) ( approximately 80%) and conjugated bis-allylic Rba(.) ( approximately 20%) radicals. The latter were detectable in the absence of oxygen by their prominent, narrow absorption band at 280 nm. The Rn(.) radicals of ArAc (containing three bis-allylic sites) transform to the Rba(.) radicals via an intramolecular H-atom abstraction [rate constant (7.5 +/- 0.7) x 10 (4) s (-1)]. In contrast, the Rn(.) radicals of LnAc that contain only one bis-allylic site do not transform intramolecularly to the Rba(.) radicals. In the case of PmAc, which contains only one double bond, the Rba(.) radicals are not observed. The Rn(.) radicals of PmAc rapidly combine with oxygen with a rate constant of (3.8 +/- 0.4) x 10(9) M(-1) s(-1). The Rba(.) radicals of ArAc are less reactive and react with oxygen with a rate constant of (2.2 +/- 0.2) x 10 (8) M (-1) s (-1). The ArAc peroxyl radicals formed spontaneously eliminate superoxide radical anions [rate constant = (3.4 +/- 0.3) x 10 (4) M (-1) s (-1)]. The stable oxidative lesions derived from the 2',3',5'-tri- O-acetylguanosine or 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine radicals and their subsequent reactions with ArAc peroxyl radicals were also investigated. The major products found were the 2,5-diamino-4 H

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

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


    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.

  8. The guanine cation radical: investigation of deprotonation states by ESR and DFT. (United States)

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


    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.

  9. Sublingual nucleotides and immune response to exercise

    Directory of Open Access Journals (Sweden)

    Ostojic Sergej M


    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.

  10. Simultaneous determination of adenine and guanine in ruminant bacterial pellets by ion-pair HPLC. (United States)

    García del Moral, Pilar; Arín, María Jesús; Resines, José Antonio; Díez, María Teresa


    An ion-pair reversed-phase high-performance liquid chromatography with gradient elution and UV detection was used to measure adenine (A) and guanine (G) in lyophilized bacterial pellets from ruminants using allopurinol as internal standard. The separation was performed on a Symmetry C18 column and the detection was monitored at 280 nm. Calibration curves were found to be linear in the concentration range from 5 to 50 mg/l with correlation coefficients (r2)>0.999. Mean recoveries of A and G standards added to bacterial samples were 102.2 and 98.2, respectively. The method proposed yielded sharp, well-resolved peaks within 25 min and was successfully applied for the determination of A and G in bacterial pellets.

  11. 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.


    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.


    Directory of Open Access Journals (Sweden)

    GIORGIO Selma


    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.

  13. Circular dichroism anisotrophy of DNA with different modifications at N7 of guanine. (United States)

    Zavriev, S K; Minchenkova, L E; Vorlícková, M; Kolchinsky, A M; Volkenstein, M V; Ivanov, V I


    The complexex DNA-Ag1+, DNA-Cu1+, protonated DNA and DNA methylated at N7 of guanine were oriented by pumping the solutions through a multicapillary cell in the direction of a light beam. The CD components along the DNA axis, delta epsilon parallel, and normal to it, 2 delta epsilon perpendicular, were calculated from the CD spectra of the oriented samples by the method of Chung and Holzwarth, (1975) J. Mol. Biol. 92, 449--466. It was shown that in most cases, except that of the protonated DNA, the degree of orientation was only slightly less than that for pure DNA. This demonstrated the absence of aggregation and of appreciable denaturation. In all cases the modifications of DNA give rise to a negative component 2 delta epsilon perpendicular, whose magnitude increased as the extent of modification increased. From both the CD spectra of non-oriented samples and the absorption spectra, an inference is drawn that Ag1+ and Cu1+ are attached to the same site as CH3 groups i.e., to the N7 atom of guanine. Proton transfer along the H-bond from the N1 atom of G to the N3 atom of the complementary cytosine is suggested to be a result of the modifications, although the case of H+-DNA may differ from the others. Based on the CD spectra for the anisotropic components, delta epsilon parallel and 2 delta epsilon perpendicular, it is proposed that ligand binding is accompanied by winding of the DNA helix.

  14. Investigation of base pairs containing oxidized guanine using ab initio method and ABEEMσπ polarizable force field. (United States)

    Liu, Cui; Wang, Yang; Zhao, Dongxia; Gong, Lidong; Yang, Zhongzhi


    The integrity of the genetic information is constantly threatened by oxidizing agents. Oxidized guanines have all been linked to different types of cancers. Theoretical approaches supplement the assorted experimental techniques, and bring new sight and opportunities to investigate the underlying microscopic mechanics. Unfortunately, there is no specific force field to DNA system including oxidized guanines. Taking high level ab initio calculations as benchmark, we developed the ABEEMσπ fluctuating charge force field, which uses multiple fluctuating charges per atom. And it was applied to study the energies, structures and mutations of base pairs containing oxidized guanines. The geometries were obtained in reference to other studies or using B3LYP/6-31+G* level optimization, which is more rational and timesaving among 24 quantum mechanical methods selected and tested by this work. The energies were determined at MP2/aug-cc-pVDZ level with BSSE corrections. Results show that the constructed potential function can accurately simulate the change of H-bond and the buckled angle formed by two base planes induced by oxidized guanine, and it provides reliable information of hydrogen bonding, stacking interaction and the mutation processes. The performance of ABEEMσπ polarizable force field in predicting the bond lengths, bond angles, dipole moments etc. is generally better than those of the common force fields. And the accuracy of ABEEMσπ PFF is close to that of the MP2 method. This shows that ABEEMσπ model is a reliable choice for further research of dynamics behavior of DNA fragment including oxidized guanine.

  15. Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing. (United States)

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E


    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

  16. Nucleotide excision repair in the test tube.

    NARCIS (Netherlands)

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


    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.

  17. Effects of nucleotides and nucleosides on coagulation

    DEFF Research Database (Denmark)

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


    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...

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

    Directory of Open Access Journals (Sweden)

    Harikrishnan Jayamohan


    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

  19. Oxidation of guanine by carbonate radicals derived from photolysis of carbonatotetramminecobalt(III) complexes and the pH dependence of intrastrand DNA cross-links mediated by guanine radical reactions. (United States)

    Crean, Conor; Lee, Young Ae; Yun, Byeong Hwa; Geacintov, Nicholas E; Shafirovich, Vladimir


    The carbonate radical anion CO(3)(*-) is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO(3)(*-) radicals is known to yield spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) products, and also a novel intrastrand cross-linked product: 5'-d(CCATCG*CT*ACC), featuring a linkage between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et al., Nucleic Acids Res. 2008, 36, 742-755). Involvement of the T-N3 (pK(a) of N3-H is 9.67) suggests that the formation of 5'-d(CCATCG*CT*ACC) might be pH-dependent. This hypothesis was tested by generating CO(3)(*-) radicals through the photodissociation of carbonatotetramminecobalt(III) complexes by steady-state UV irradiation, which allowed for studies of product yields in the pH 5.0-10.0 range. The yield of 5'-d(CCATCG*CT*ACC) at pH 10.0 is approximately 45 times greater than at pH 5.0; this is consistent with the proposed mechanism, which requires N3(H) thymine proton dissociation followed by nucleophilic addition to the C8 guanine radical.

  20. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process. (United States)

    Yang, C. C.; Oro, J.


    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).

  1. Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine. (United States)

    Grimme, Stefan; Bauer, Christopher Alexander


    The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

  2. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis. (United States)

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


    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 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.

  3. Quantum-chemical study of interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-nucleobases. (United States)

    Mikulski, Damian; Szeląg, Małgorzata; Molski, Marcin


    Trans-resveratrol, a natural phytoalexin present in red wine and grapes, has gained considerable attention because of its antiproliferative, chemopreventive and proapoptotic activity against human cancer cells. The accurate quantum-chemical computations based on the density functional theory (DFT) and ab initio second-order Møller-Plesset perturbation method (MP2) have been performed for the first time to study interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-derived nitrogenous bases: adenine, guanine, cytosine and thymine in vacuum and water medium. This compound is found to show high affinity to nitrogenous bases and guanine-thymine dinucleotide. The electrostatic interactions from intermolecular hydrogen bonding increase the stability of complexes studied. In particular, significantly strong hydrogen bonds between 4'-H atom of trans-resveratrol and imidazole nitrogen as well as carbonyl oxygen atoms of nucleobases studied stabilize these systems. The stabilization energies computed reveal that the negatively charged trans-resveratrol-dinucleotide complex is more energetically stable in water medium than in vacuum. MP2 method gives more reliable and significantly high values of stabilization energy of trans-resveratrol-dinucleotide, trans-resveratrol-guanine and trans-resveratrol-thymine complexes than B3LYP exchange-correlation functional because it takes into account London dispersion energy. According to the results, in the presence of trans-resveratrol the 3'-5' phosphodiester bond in dinucleotide can be cleaved and the proton from 4'-OH group of trans-resveratrol migrates to the 3'-O atom of dinucleotide. It is concluded that trans-resveratrol is able to break the DNA strand. Hence, the findings obtained help understand antiproliferative and anticancer properties of this polyphenol.

  4. ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions. (United States)

    Levy, Michael A; Kernohan, Kristin D; Jiang, Yan; Bérubé, Nathalie G


    ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.

  5. Structural and energetic heterogeneities of canonical and oxidized central guanine triad of B-DNA telomeric fragments. (United States)

    Cysewski, Piotr; Czeleń, Przemysław


    The intermolecular interaction energies in central guanine triad of telomeric B-DNA were estimated based on ab initio quantum chemistry calculations on the MP2/aDZ level of theory. The source of structural information was molecular dynamics simulation of both canonical (AGGGTT) and oxidized (AG8oxoGGTT) telomere units. Our calculations demonstrate that significant stiffness of central triad occurs if 8oxoG is present. The origin of such feature is mainly due to the increase of stacking interactions of 8oxoG with neighbouring guanine molecules and stronger hydrogen bonding formation of 8oxoG with cytosine if compared with canonical guanine. Another interesting observation is the context independence of stacking interactions of 8oxoG. Unlike to 5'-G2/G3-3' and 5'-G3/G4-3' sequences which are energetically different, 5'-G2/8oxoG3-3' and 5'-8oxoG3/G4-3' sequences are almost iso-energetic.

  6. Sequence-selective binding of phenazinium dyes phenosafranin and safranin O to guanine-cytosine deoxyribopolynucleotides: spectroscopic and thermodynamic studies. (United States)

    Saha, Ishita; Hossain, Maidul; Suresh Kumar, Gopinatha


    The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 10(5) M(-1), but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA)·poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O.

  7. Comparison of the acid-base properties of ribose and 2'-deoxyribose nucleotides. (United States)

    Mucha, Ariel; Knobloch, Bernd; Jezowska-Bojczuk, Małgorzata; Kozłowski, Henryk; Sigel, Roland K O


    The extent to which the replacement of a ribose unit by a 2'-deoxyribose unit influences the acid-base properties of nucleotides has not hitherto been determined in detail. In this study, by potentiometric pH titrations in aqueous solution, we have measured the acidity constants of the 5'-di- and 5'-triphosphates of 2'-deoxyguanosine [i.e., of H(2)(dGDP)(-) and H(2)(dGTP)(2-)] as well as of the 5'-mono-, 5'-di-, and 5'-triphosphates of 2'-deoxyadenosine [i.e., of H(2)(dAMP)(+/-), H(2)(dADP)(-), and H(2)(dATP)(2-)]. These 12 acidity constants (of the 56 that are listed) are compared with those of the corresponding ribose derivatives (published data) measured under the same experimental conditions. The results show that all protonation sites in the 2'-deoxynucleotides are more basic than those in their ribose counterparts. The influence of the 2'-OH group is dependent on the number of 5'-phosphate groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is most significantly enhanced (by about 0.2 pK units), while the effect on the phosphate groups and the N1H or N1H(+) sites is less pronounced but clearly present. In addition, (1)H NMR chemical shift change studies in dependence on pD in D(2)O have been carried out for the dAMP, dADP, and dATP systems, which confirmed the results from the potentiometric pH titrations and showed the nucleotides to be in their anti conformations. Overall, our results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact basis for the calculation, determination, and understanding of perturbed pK(a) values in DNAzymes and ribozymes, as needed for the delineation of acid-base mechanisms in catalysis.

  8. Quadruplex-single nucleotide polymorphisms (Quad-SNP) influence gene expression difference among individuals. (United States)

    Baral, Aradhita; Kumar, Pankaj; Halder, Rashi; Mani, Prithvi; Yadav, Vinod Kumar; Singh, Ankita; Das, Swapan K; Chowdhury, Shantanu


    Non-canonical guanine quadruplex structures are not only predominant but also conserved among bacterial and mammalian promoters. Moreover recent findings directly implicate quadruplex structures in transcription. These argue for an intrinsic role of the structural motif and thereby posit that single nucleotide polymorphisms (SNP) that compromise the quadruplex architecture could influence function. To test this, we analysed SNPs within quadruplex motifs (Quad-SNP) and gene expression in 270 individuals across four populations (HapMap) representing more than 14,500 genotypes. Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P analysis of Quad-SNPs obtained from population-scale sequencing of 1000 human genomes showed relative selection bias against alteration of the structural motif. To directly test the quadruplex-SNP-transcription connection, we constructed a reporter system using the RPS3 promoter-remarkable difference in promoter activity in the 'quadruplex-destabilized' versus 'quadruplex-intact' promoter was noticed. As a further test, we incorporated a quadruplex motif or its disrupted counterpart within a synthetic promoter reporter construct. The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin. Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals.

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

    NARCIS (Netherlands)

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


    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

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


    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.

  11. The International Nucleotide Sequence Database Collaboration (United States)

    Cochrane, Guy; Karsch-Mizrachi, Ilene; Takagi, Toshihisa; Sequence Database Collaboration, International Nucleotide


    The International Nucleotide Sequence Database Collaboration (INSDC; comprises three global partners committed to capturing, preserving and providing comprehensive public-domain nucleotide sequence information. The INSDC establishes standards, formats and protocols for data and metadata to make it easier for individuals and organisations to submit their nucleotide data reliably to public archives. This work enables the continuous, global exchange of information about living things. Here we present an update of the INSDC in 2015, including data growth and diversification, new standards and requirements by publishers for authors to submit their data to the public archives. The INSDC serves as a model for data sharing in the life sciences. PMID:26657633

  12. 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


    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.

  13. Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry. (United States)

    Ding, Xi-Qin; Matveev, Alexander; Singh, Anil; Komori, Naoka; Matsumoto, Hiroyuki


    Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.

  14. Effects of nucleotides and nucleosides on coagulation

    DEFF Research Database (Denmark)

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


    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......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.......7 ng/ml; P blood was evaluated by TEG. Circulating ADP induces hypocoagulation without signs of increased fibrinolysis as evaluated by TEG. The potential...

  15. Identification of aflatoxin M1-N7-guanine in liver and urine of tree shrews and rats following administration of aflatoxin B1. (United States)

    Egner, Patricia A; Yu, Xiang; Johnson, Jesse K; Nathasingh, Christopher K; Groopman, John D; Kensler, Thomas W; Roebuck, Bill D


    Epidemiological studies have shown that exposure to aflatoxin B(1) (AFB(1)) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemical-viral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB(1) in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M(1)-N(7)-guanine (AFM(1)-N(7)-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 microg/kg AFB(1). In hepatic DNA isolated from tree shrews, AFM(1)-N(7)-guanine was the predominant adduct, 0.74 +/- 0.14 pmol/mg DNA, as compared to 0.37 +/- 0.07 pmol/mg DNA of AFB(1)-N(7)-guanine. Conversely, in rat liver, 6.56 +/- 2.41 pmol/mg DNA of AFB(1)-N(7)-guanine and 0.42 +/- 0.13 pmol/mg DNA of AFM(1)-N(7)-guanine were detected. Rats excreted 1.00 +/- 0.21 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.29 +/- 0.10 pmol AFM(1)-N(7)-guanine/mg creatinine as compared to 0.60 +/- 0.12 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.69 +/- 0.16 pmol AFM(1)-N(7)-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM(1), than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q(1) or aflatoxin P(1) but formed a significantly greater amount of AFM(1) than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB(1) hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.

  16. 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)


    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.

  17. 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


    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.

  18. [Sublicons containing amino acids and nucleotides]. (United States)

    Kaĭmakov, E A


    Sublicons have been obtained. Sublicons are threadlike structures appearing during sublimation of frozen solutions of small concentrations, containing racemate mixture of amino acids and nucleotides. It is suggested that close location of chains and their zonal distribution by the section of helix spire forming sublicon wall, should provide the formation of stereohomogenous and complementary successions of biomonomers of different clases.

  19. 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.

  20. 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. (United States)

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


    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.

  1. The Role of Aspartic Acid 143 in E. coli tRNA-Guanine Transglycosylase: Insights from Mutagenesis Studies and Computational Modeling (United States)

    Todorov, Katherine Abold; Tan, Xiao-Jian; Nonekowski, Susanne T.; Garcia, George A.; Carlson, Heather A.


    tRNA guanine transglycosylase (TGT) is a tRNA-modifying enzyme which catalyzes the posttranscriptional exchange of guanine in position 34 of tRNAY,H,N,D with the modified base queuine in eukaryotes or its precursor, preQ1 base, in eubacteria. Thus, TGT must recognize the guanine in tRNA and the free base queuine or preQ1 to catalyze this exchange. The crystal structure of Zymomonas mobilis TGT with preQ1 bound suggests that a key aspartate is critically involved in substrate recognition. To explore this, a series of site-directed mutants of D143 in Escherichia coli TGT were made and characterized to investigate heterocyclic substrate recognition. Our data confirm that D143 has significant impact on KM of guanine; however, the trend in the KM data (D143A D143A > D143N > D143S > D143T appears to be directly related to the degree of hydrogen bonding available to guanine in the binding site. PMID:15951383

  2. Preparation of a sol-gel-derived carbon nanotube ceramic electrode by microwave irradiation and its application for the determination of adenine and guanine

    Energy Technology Data Exchange (ETDEWEB)

    Abbaspour, Abdolkarim, E-mail: [Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Fars 71456-85464 (Iran, Islamic Republic of); Ghaffarinejad, Ali [Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Fars 71456-85464 (Iran, Islamic Republic of)


    In this study, microwave irradiation was used for the fast preparation (min) of a sol-gel-derived carbon nanotube ceramic electrode (MW-CNCE). For confirmation of the preparation of the ceramic by MW irradiation, Fourier transform infrared, X-ray diffraction spectra and scanning electron microscopy images of the produced ceramic were compared with those of conventional ceramic (which is produced by drying the ceramic in air for 48 h). The electrochemical behavior of MW-CNCE in nicotinamide adenine dinucleotide, L-cysteine, adenine and guanine was compared with that of a conventional sol-gel-derived carbon nanotube ceramic electrode (CNCE). In all systems, similar peak potentials and lower background currents were obtained with respect to CNCE. Finally, the MW-CNCE was used for the simultaneous determination of adenine and guanine using differential pulse voltammetry. The linear ranges of 0.1-10 and 0.1-20 muM were obtained for adenine and guanine, respectively. These results are comparable with some modified electrodes that have recently been reported for the determination of adenine and guanine, with the advantage that the proposed electrode did not contain modifier. In addition, the proposed electrode was successfully used for the oxidation of adenine and guanine in DNA, and the detection limit for this measurement was 0.05 mug mL{sup -1} DNA.

  3. 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.


    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.

  4. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Joonghoon Park

    Full Text Available Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS are important signaling molecules for embryonic stem cell (ESC differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG. Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-2ME than (+2ME group (p < 0.05. This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05 and increased formation of beating embryoid bodies (EBs; p < 0.001. Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-2ME (p < 0.01. Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative

  5. The electrochemical reduction of the purines guanine and adenine at platinum electrodes in several room temperature ionic liquids

    Energy Technology Data Exchange (ETDEWEB)

    Zanoni, Maria Valnice Boldrin, E-mail: [Department of Analytical Chemistry, Institute of Chemistry, University of Sao Paulo State, Araraquara, R. Prof. Francisco Degni, CP 355, 14801-970, SP (Brazil); Rogers, Emma I. [Department of Chemistry, Physical and Theoretical Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QZ (United Kingdom); Hardacre, Christopher, E-mail: [School of Chemistry and Chemical Engineering/QUILL, Queen' s University Belfast, Belfast, Northern Ireland BT9 5AG (United Kingdom); Compton, Richard G., E-mail: [Department of Chemistry, Physical and Theoretical Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QZ (United Kingdom)


    The reduction of guanine was studied by microelectrode voltammetry in the room temperature ionic liquids (RTILs) N-hexyltriethylammonium bis (trifluoromethanesulfonyl) imide [N{sub 6,2,2,2}][N(Tf){sub 2}], 1-butyl-3-methylimidazolium hexafluorosphosphate [C{sub 4}mim][PF{sub 6}], N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide [C{sub 4}mpyrr][N(Tf){sub 2}], 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C{sub 4}mim][N(Tf){sub 2}], N-butyl-N-methyl-pyrrolidinium dicyanamide [C{sub 4}mpyrr][N(NC){sub 2}] and tris(P-hexyl)-tetradecylphosphonium trifluorotris(pentafluoroethyl)phosphate [P{sub 14,6,6,6}][FAP] on a platinum microelectrode. In [N{sub 6,2,2,2}][NTf{sub 2}] and [P{sub 14,6,6,6}][FAP], but not in the other ionic liquids studied, guanine reduction involves a one-electron, diffusion-controlled process at very negative potential to produce an unstable radical anion, which is thought to undergo a dimerization reaction, probably after proton abstraction from the cation of the ionic liquid. The rate of this subsequent reaction depends on the nature of the ionic liquid, and it is faster in the ionic liquid [P{sub 14,6,6,6}][FAP], in which the formation of the resulting dimer can be voltammetrically monitored at less negative potentials than required for the reduction of the parent molecule. Adenine showed similar behaviour to guanine but the pyrimidines thymine and cytosine did not; thymine was not reduced at potentials less negative than required for solvent (RTIL) decomposition while only a poorly defined wave was seen for cytosine. The possibility for proton abstraction from the cation in [N{sub 6,2,2,2}][NTf{sub 2}] and [P{sub 14,6,6,6}][FAP] is noted and this is thought to aid the electrochemical dimerization process. The resulting rapid reaction is thought to shift the reduction potentials for guanine and adenine to lower values than observed in RTILs where the scope for proton abstraction is not present. Such shifts are

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

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


    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.

  7. 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)


    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.

  8. Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa (United States)

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


    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

  9. The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG. (United States)

    Aurandt, Jennifer; Vikis, Haris G; Gutkind, J Silvio; Ahn, Natalie; Guan, Kun-Liang


    Semaphorins are axon guidance molecules that signal through the plexin family of receptors. Semaphorins also play a role in other processes such as immune regulation and tumorigenesis. However, the molecular signaling mechanisms downstream of plexin receptors have not been elucidated. Semaphorin 4D is the ligand for the plexin-B1 receptor and stimulation of the plexin-B1 receptor activates the small GTPase RhoA. Using the intracellular domain of plexin-B1 as an affinity ligand, two Rho-specific guanine nucleotide exchange factors, leukemia-associated Rho GEF (LARG; GEF, guanine nucleotide exchange factors) and PSD-95/Dlg/ZO-1 homology (PDZ)-RhoGEF, were isolated from mouse brain as plexin-B1-specific interacting proteins. LARG and PDZ-RhoGEF contain several functional domains, including a PDZ domain. Biochemical characterizations showed that the PDZ domain of LARG is directly involved in the interaction with the carboxy-terminal sequence of plexin-B1. Mutation of either the PDZ domain in LARG or the PDZ binding site in plexin-B1 eliminates the interaction. The interaction between plexin-B1 and LARG is specific for the PDZ domain of LARG and LARG does not interact with plexin-A1. A LARG-interaction defective mutant of the plexin-B1 receptor was created and was unable to stimulate RhoA activation. The data in this report suggest that LARG plays a critical role in plexin-B1 signaling to stimulate Rho activation and cytoskeletal reorganization.

  10. Radiation and thermal stabilities of adenine nucleotides. (United States)

    Demidov, V V; Potaman, V N; Solyanina, I P; Trofimov, V I


    We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

  11. Nucleotide sequence of papaya mosaic virus RNA. (United States)

    Sit, T L; Abouhaidar, M G; Holy, S


    The RNA genome of papaya mosaic virus is 6656 nucleotides long [excluding the poly(A) tail] with six open reading frames (ORFs) more than 200 nucleotides long. The four nearest the 5' end each overlap with adjacent ORFs and could code for proteins with Mr 176307, 26248, 11949 and 7224 (ORFs 1 to 4). The fifth ORF produces the capsid protein of Mr 23043 and the sixth ORF, located completely within ORF1, could code for a protein with Mr 14113. The translation products of ORFs 1 to 3 show strong similarity with those of other potexviruses but the ORF 4 protein has only limited similarity with the other potexvirus ORF 4 proteins of 7K to 11K.

  12. Multiphasic interactions between nucleotides and target proteins

    CERN Document Server

    Nissen, Per


    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 ...

  13. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Joong-Won, E-mail: [Division of Science, Governors State University, University Park, Illinois 60484-0975 (United States); Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States); Bernstein, Elliot R., E-mail: [Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States)


    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  14. Visualization of cyclic nucleotide dynamics in neurons

    Directory of Open Access Journals (Sweden)

    Kirill eGorshkov


    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.

  15. Constructing a novel 8-hydroxy-2'-deoxyguanosine electrochemical sensor and application in evaluating the oxidative damages of DNA and guanine. (United States)

    Guo, Zhipan; Liu, Xiuhui; Liu, Yuelin; Wu, Guofan; Lu, Xiaoquan


    8-Hydroxy-2'-deoxyguanosine (8-OHdG) is commonly identified as a biomarker of oxidative DNA damage. In this work, a novel and facile 8-OHdG sensor was developed based on the multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE). It exhibited good electrochemical responses toward the oxidation of 8-OHdG, and the linear ranges were 5.63×10(-8)-6.08×10(-6)M and 6.08×10(-6)-1.64×10(-5)M, with the detection limit of 1.88×10(-8)M (S/N=3). Moreover, the fabricated sensor was applied for the determination of 8-OHdG generated from damaged DNA and guanine, respectively, and the oxidation currents of 8-OHdG increased along with the damaged DNA and guanine within certain concentrations. These results could be used to evaluate the DNA damage, and provide useful information on diagnosing diseases caused by mutation and deficiency of the immunity system.

  16. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity. (United States)

    Todorov, Katherine Abold; Garcia, George A


    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.

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

    KAUST Repository

    Chawla, Mohit


    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).

  18. Quadruplexes of human telomere dG{sub 3}(TTAG{sub 3}){sub 3} sequences containing guanine abasic sites

    Energy Technology Data Exchange (ETDEWEB)

    Skolakova, Petra; Bednarova, Klara; Vorlickova, Michaela [Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno (Czech Republic); Sagi, Janos, E-mail: [Rimstone Laboratory, RLI, 29 Lancaster Way, Cheshire, CT 06410 (United States)


    Research highlights: {yields} Loss of a guanine base does not hinder the formation of G-quadruplex of human telomere sequence. {yields} Each depurination strongly destabilizes the quadruplex of dG{sub 3}(TTAG{sub 3}){sub 3} in NaCl and KCl. {yields} Conformational change of the abasic analogs of dG{sub 3}(TTAG{sub 3}){sub 3} is inhibited in KCl. {yields} The effects abasic sites may affect telomere-end structures in vivo. -- Abstract: This study was performed to evaluate how the loss of a guanine base affects the structure and stability of the three-tetrad G-quadruplex of 5'-dG{sub 3}(TTAG{sub 3}){sub 3}, the basic quadruplex-forming unit of the human telomere DNA. None of the 12 possible abasic sites hindered the formation of quadruplexes, but all reduced the thermodynamic stability of the parent quadruplex in both NaCl and KCl. The base loss did not change the Na{sup +}-stabilized intramolecular antiparallel architecture, based on CD spectra, but held up the conformational change induced in dG{sub 3}(TTAG{sub 3}){sub 3} in physiological concentration of KCl. The reduced stability and the inhibited conformational transitions observed here in vitro for the first time may predict that unrepaired abasic sites in G-quadruplexes could lead to changes in the chromosome's terminal protection in vivo.

  19. The mitochondrial genome sequence of the ciliate Paramecium caudatum reveals a shift in nucleotide composition and codon usage within the genus Paramecium

    Directory of Open Access Journals (Sweden)

    Berendonk Thomas U


    Full Text Available Abstract Background Despite the fact that the organization of the ciliate mitochondrial genome is exceptional, only few ciliate mitochondrial genomes have been sequenced until today. All ciliate mitochondrial genomes are linear. They are 40 kb to 47 kb long and contain some 50 tightly packed genes without introns. Earlier studies documented that the mitochondrial guanine + cytosine contents are very different between Paramecium tetraurelia and all studied Tetrahymena species. This raises the question of whether the high mitochondrial G+C content observed in P. tetraurelia is a characteristic property of Paramecium mtDNA, or whether it is an exception of the ciliate mitochondrial genomes known so far. To test this question, we determined the mitochondrial genome sequence of Paramecium caudatum and compared the gene content and sequence properties to the closely related P. tetraurelia. Results The guanine + cytosine content of the P. caudatum mitochondrial genome was significantly lower than that of P. tetraurelia (22.4% vs. 41.2%. This difference in the mitochondrial nucleotide composition was accompanied by significantly different codon usage patterns in both species, i.e. within P. caudatum clearly A/T ending codons dominated, whereas for P. tetraurelia the synonymous codons were more balanced with a higher number of G/C ending codons. Further analyses indicated that the nucleotide composition of most members of the genus Paramecium resembles that of P. caudatum and that the shift observed in P. tetraurelia is restricted to the P. aurelia species complex. Conclusions Surprisingly, the codon usage bias in the P. caudatum mitochondrial genome, exemplified by the effective number of codons, is more similar to the distantly related T. pyriformis and other single-celled eukaryotes such as Chlamydomonas, than to the closely related P. tetraurelia. These differences in base composition and codon usage bias were, however, not reflected in the amino

  20. A highly specific and sensitive electroanalytical strategy for microRNAs based on amplified silver deposition by the synergic TiO2 photocatalysis and guanine photoreduction using charge-neutral probes. (United States)

    Li, Rui; Li, Shuying; Dong, Minmin; Zhang, Liyan; Qiao, Yuchun; Jiang, Yao; Qi, Wei; Wang, Hua


    TiO2 photocatalysis and guanine photoreduction were synergically combined for amplifying silver deposition for the electroanalysis of short-chain microRNAs with guanine bases using charge-neutral probes. It could allow for the highly specific and sensitive detection of microRNAs in the blood as well as the identification of their mutant levels.

  1. Histone displacement during nucleotide excision repair

    DEFF Research Database (Denmark)

    Dinant, C.; Bartek, J.; Bekker-Jensen, S.


    Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER. © 2012 by the authors; licensee MDPI, Basel...

  2. In Vitro Selection Using Modified or Unnatural Nucleotides (United States)

    Stovall, Gwendolyn M.; Bedenbaugh, Robert S.; Singh, Shruti; Meyer, Adam J.; Hatala, Paul J.; Ellington, Andrew D.; Hall, Bradley


    Incorporation of modified nucleotides into in vitro RNA or DNA selections offer many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions. PMID:25606981

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

    Directory of Open Access Journals (Sweden)

    Dong L


    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

  4. Evaluation of the flanking nucleotide sequences of sarcomeric hypertrophic cardiomyopathy substitution mutations. (United States)

    Meurs, Kathryn M; Mealey, Katrina L


    Hypertrophic cardiomyopathy (HCM) is a familial myocardial disease with a prevalence of 1 in 500. More than 400 causative mutations have been identified in 13 sarcomeric and myofilament related genes, 350 of these are substitution mutations within eight sarcomeric genes. Within a population, examples of recurring identical disease causing mutations that appear to have arisen independently have been noted as well as those that appear to have been inherited from a common ancestor. The large number of novel HCM mutations could suggest a mechanism of increased mutability within the sarcomeric genes. The objective of this study was to evaluate the most commonly reported HCM genes, beta myosin heavy chain (MYH7), myosin binding protein C, troponin I, troponin T, cardiac regulatory myosin light chain, cardiac essential myosin light chain, alpha tropomyosin and cardiac alpha-actin for sequence patterns surrounding the substitution mutations that may suggest a mechanism of increased mutability. The mutations as well as the 10 flanking nucleotides were evaluated for frequency of di-, tri- and tetranucleotides containing the mutation as well as for the presence of certain tri- and tetranculeotide motifs. The most common substitutions were guanine (G) to adenine (A) and cytosine (C) to thymidine (T). The CG dinucleotide had a significantly higher relative mutability than any other dinucleotide (pmutation was calculated; none were at a statistically higher frequency than the others. The large number of G to A and C to T mutations as well as the relative mutability of CG may suggest that deamination of methylated CpG is an important mechanism for mutation development in at least some of these cardiac genes.

  5. Estimation of evolutionary distances between nucleotide sequences. (United States)

    Zharkikh, A


    A formal mathematical analysis of the substitution process in nucleotide sequence evolution was done in terms of the Markov process. By using matrix algebra theory, the theoretical foundation of Barry and Hartigan's (Stat. Sci. 2:191-210, 1987) and Lanave et al.'s (J. Mol. Evol. 20:86-93, 1984) methods was provided. Extensive computer simulation was used to compare the accuracy and effectiveness of various methods for estimating the evolutionary distance between two nucleotide sequences. It was shown that the multiparameter methods of Lanave et al.'s (J. Mol. Evol. 20:86-93, 1984), Gojobori et al.'s (J. Mol. Evol. 18:414-422, 1982), and Barry and Hartigan's (Stat. Sci. 2:191-210, 1987) are preferable to others for the purpose of phylogenetic analysis when the sequences are long. However, when sequences are short and the evolutionary distance is large, Tajima and Nei's (Mol. Biol. Evol. 1:269-285, 1984) method is superior to others.

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

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


    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.

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

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


    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.

  8. Two-step enzymatic synthesis of guanine arabionside%两步酶法合成阿糖鸟苷

    Institute of Scientific and Technical Information of China (English)

    魏晓琨; 丁庆豹; 欧伶; 张鹭; 王昌禄


    The chemical synthesis of Guanine arabinoside (ara-G) is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside (ara-DA) was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phos-phorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L-1 uracil arabinoside as arabinose donor, 10 mmol·L-1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L-1 phosphate buffer, and reacted at 60℃ for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.

  9. Fluorescence properties of 8-(2-pyridyl)guanine "2PyG" as compared to 2-aminopurine in DNA. (United States)

    Dumas, Anaëlle; Luedtke, Nathan W


    Because of their environment-sensitive fluorescence quantum yields, base analogues such as 2-aminopurine (2AP), 6-methylisoxanthopterin (6-MI), and 3-methylisoxanthopterin (3-MI) are widely used in nucleic-acid folding and catalysis assays. Emissions from these guanine mimics are quenched by base-stacking interactions and collisions with purine residues. Fluorescent base analogues that remain highly emissive in folded nucleic acids can provide sensitive means to differentiate DNA/RNA structures by participating in energy transfer from proximal ensembles of unmodified nucleobases. The development of new, highly emissive guanine mimics capable of proper base stacking and base-pairing interactions is an important prerequisite to this approach. Here we report a comparison of the most commonly used probe, 2-aminopurine (2AP), to 8-(2-pyridyl)-2'-deoxyguanosine (2PyG). The photophysical properties of these purine derivatives are very different. 2PyG exhibits enhanced fluorescence quantum yields upon its incorporation into folded nucleic acids--approximately 50-fold brighter fluorescence intensity than 2AP in the context of duplex DNA. Due to its bright fluorescence and compatibility with proper DNA folding, 2PyG can be used to accurately quantify energy-transfer efficiencies, whereas 2AP is much less sensitive to structure-specific trends in energy transfer. When using nucleoside monomers, Stern-Volmer plots of 2AP fluorescence revealed upward curvature of F(0) /F upon titration of guanosine monophoshate (GMP), whereas 2PyG exhibited unusual downward curvature of F(0) /F that resulted in a recovery of fluorescence at high GMP concentrations. These results are consistent with the trends observed for 2PyG- and 2AP-containing oligonucleotides, and furthermore suggest that solutions containing high concentrations of GMP can, in some ways, mimic the high local nucleobase densities of folded nucleic acids.

  10. New Dihydro OO'Bis(Salicylidene 2,2' Aminobenzothiazolyl Borate Complexes: Kinetic and Voltammetric Studies of Dimethyltin Copper Complex with Guanine, Adenine, and Calf Thymus DNA

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    Full Text Available The newly synthesized ligand, dihydro OO'bis(salicylidene 2,2' aminobenzothiazolyl borate (2, was derived from the reaction of Schiff base of 2-aminobenzothiazole and salicylaldehyde with KBH 4 . Cu II (3 and Zn II (4 complexes of (2 were synthesized and further metallated with dimethyltindichloride to yield heterobimetallic complexes (5 and (6. All complexes have been thoroughly characterized by elemental analysis, and IR, NMR, EPR, and UV-Vis spectroscopy and conductance measurements. The spectroscopic data support square planar environment around the Cu II atom, while the Sn IV atom acquires pentacoordinate geometry. The interaction of complex (5 with guanine, adenine, and calf thymus DNA was studied by spectrophotometric, electrochemical, and kinetic methods. The absorption spectra of complex (5 exhibit a remarkable "hyperchromic effect" in the presence of guanine and calf thymus DNA. Indicative of strong binding of the complex to calf thymus DNA preferentially binds through N 7 position of guanine base, while the adenine shows binding to a lesser extent. The kinetic data were obtained from the rate constants, k obs , values under pseudo-first-order conditions. Cyclic voltammetry was employed to study the interaction of complex (5 with guanine, adenine, and calf thymus DNA. The CV of complex (5 in the absence and in the presence of guanine and calf thymus DNA altered drastically, with a positive shift in formal peak potential E pa and E pc values and a significant increase in peak current. The positive shift in formal potentials with increase in peak current favours strong interaction of complex (5 with calf thymus DNA. The net shift in E 1/2 has been used to estimate the ratio of equilibrium constants for the binding of Cu(II and Cu(I complexes to calf thymus DNA.

  11. New Dihydro OO'Bis(Salicylidene) 2,2' Aminobenzothiazolyl Borate Complexes: Kinetic and Voltammetric Studies of Dimethyltin Copper Complex with Guanine, Adenine, and Calf Thymus DNA. (United States)

    Arjmand, Farukh; Mohani, Bhawana; Parveen, Shamima


    The newly synthesized ligand, dihydro OO'bis(salicylidene) 2,2' aminobenzothiazolyl borate (2), was derived from the reaction of Schiff base of 2-aminobenzothiazole and salicylaldehyde with KBH(4). Cu(II) (3) and Zn(II) (4) complexes of (2) were synthesized and further metallated with dimethyltindichloride to yield heterobimetallic complexes (5) and (6). All complexes have been thoroughly characterized by elemental analysis, and IR, NMR, EPR, and UV-Vis spectroscopy and conductance measurements. The spectroscopic data support square planar environment around the Cu(II) atom, while the Sn(IV) atom acquires pentacoordinate geometry. The interaction of complex (5) with guanine, adenine, and calf thymus DNA was studied by spectrophotometric, electrochemical, and kinetic methods. The absorption spectra of complex (5) exhibit a remarkable "hyperchromic effect" in the presence of guanine and calf thymus DNA. Indicative of strong binding of the complex to calf thymus DNA preferentially binds through N(7) position of guanine base, while the adenine shows binding to a lesser extent. The kinetic data were obtained from the rate constants, k(obs), values under pseudo-first-order conditions. Cyclic voltammetry was employed to study the interaction of complex (5) with guanine, adenine, and calf thymus DNA. The CV of complex (5) in the absence and in the presence of guanine and calf thymus DNA altered drastically, with a positive shift in formal peak potential E(pa) and E(pc) values and a significant increase in peak current. The positive shift in formal potentials with increase in peak current favours strong interaction of complex (5) with calf thymus DNA. The net shift in E(1/2) has been used to estimate the ratio of equilibrium constants for the binding of Cu(II) and Cu(I) complexes to calf thymus DNA.

  12. Performance characteristics of guanine incorporated PVDF-HFP/PEO polymer blend electrolytes with binary iodide salts for dye-sensitized solar cells (United States)

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


    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.

  13. A novel nuclear role for the Vav3 nucleotide exchange factor in androgen receptor coactivation in prostate cancer. (United States)

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


    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.

  14. GS-9219/VDC-1101 - a prodrug of the acyclic nucleotide PMEG has antitumor activity inspontaneous canine multiple myeloma (United States)


    Background Multiple myeloma (MM) is an important human and canine cancer for which novel therapies remain necessary. VDC-1101 (formerly GS-9219), a novel double prodrug of the anti-proliferative nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine (PMEG), possesses potent cytotoxic activity in vitro in human lymphoblasts and leukemia cell lines and in vivo in spontaneous canine lymphoma. Given the similarity in lineage between lymphoma and MM, we hypothesized that VDC-1101 would be active against MM. Results We evaluated the in vitro antiproliferative effects of VDC-1101 against 3 human MM cell lines, and we performed a phase-II clinical trial in 14 dogs with spontaneous MM. Each dog was treated with a maximum of 6 doses of VDC-1101 monotherapy over 10–15 weeks. Dose-dependent antiproliferative activity was observed in all evaluated cell lines. Major antitumor responses (reduction of serum paraprotein and resolution of hypercalcemia, peripheral cytopenias and bone marrow plasmacytosis) were observed in 9 of 11 evaluable dogs for a median of 172 days, including a durable stringent complete response (>1047 days) in a dog with melphalan-refractory disease. 2 dogs were euthanized due to presumed pulmonary fibrosis; there were no other dose-limiting toxicities encountered. Conclusions In conclusion, VDC-1101 has significant anti-tumor activity at well-tolerated doses in spontaneous canine MM. PMID:24460928

  15. On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF

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    Bushweller John H


    Full Text Available Abstract Background The Dbl-family of guanine nucleotide exchange factors (GEFs activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like domain, act as effectors of select GPCRs via Gα12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG. Results Here we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition. Conclusion Our results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.

  16. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao


    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.

  17. Fluoride ion promoted deprotection and transesterification in nucleotide triesters. (United States)

    Ogilvie, K K; Beaucage, S L


    Tetrabutylammonium fluoride will remove phenyl, trichloroethyl and cyanoethyl groups from nucleotides. In addition to the desired nucleotide products other results including chain cleavage, phosphofluoridates and cyanoethylated thymidine units may be obtained depending on the conditions used. Fluoride ion has been used to successfully exchange phenyl and trichloroethyl groups for methyl, ethyl and butyl groups in nucleotide triesters. This represents a rapid high yield route to a variety of phosphate esters. The synthesis of a novel nucleotide analogue in which two chains are bridged through their phosphates is described.

  18. Frequency and Correlation of Nearest Neighboring Nucleotides in Human Genome (United States)

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


    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.

  19. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals. (United States)

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail


    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.

  20. Cyclic nucleotide specific phosphodiesterases of Leishmania major

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    Linder Markus


    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

  1. Prebiotic nucleotide synthesis demonstration of a geologically plausible pathway

    NARCIS (Netherlands)

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


    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

  2. Nucleotide Excision Repair in Caenorhabditis elegans

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    Hannes Lans


    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.

  3. Human molecular cytogenetics: From cells to nucleotides. (United States)

    Riegel, Mariluce


    The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnormalities and the evolution of chromosomes. The development of fluorescent molecules that either directly or via an intermediate molecule bind to DNA has led to the development of fluorescent in situ hybridization (FISH), a technology linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the dimension of chromosome analysis. The field of cytogenetics is particularly important for medical diagnostics and research as well as for gene ordering and mapping. Furthermore, the increased application of molecular biology techniques, such as array-based technologies, has led to improved resolution, extending the recognized range of microdeletion/microduplication syndromes and genomic disorders. In adopting these newly expanded methods, cytogeneticists have used a range of technologies to study the association between visible chromosome rearrangements and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable number of potential applications, ranging from physical mapping to clinical and evolutionary studies, making a powerful and informative complement to other molecular and genomic approaches. This manuscript does not present a detailed history of the development of molecular cytogenetics; however, references to historical reviews and experiments have been provided whenever possible. Herein, the basic principles of molecular cytogenetics, the technologies used to identify chromosomal rearrangements and copy number changes, and the applications for cytogenetics in biomedical diagnosis and research are presented and discussed.

  4. Human molecular cytogenetics: from cells to nucleotides

    Directory of Open Access Journals (Sweden)

    Mariluce Riegel


    Full Text Available The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnormalities and the evolution of chromosomes. The development of fluorescent molecules that either directly or via an intermediate molecule bind to DNA has led to the development of fluorescent in situ hybridization (FISH, a technology linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the dimension of chromosome analysis. The field of cytogenetics is particularly important for medical diagnostics and research as well as for gene ordering and mapping. Furthermore, the increased application of molecular biology techniques, such as array-based technologies, has led to improved resolution, extending the recognized range of microdeletion/microduplication syndromes and genomic disorders. In adopting these newly expanded methods, cytogeneticists have used a range of technologies to study the association between visible chromosome rearrangements and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable number of potential applications, ranging from physical mapping to clinical and evolutionary studies, making a powerful and informative complement to other molecular and genomic approaches. This manuscript does not present a detailed history of the development of molecular cytogenetics; however, references to historical reviews and experiments have been provided whenever possible. Herein, the basic principles of molecular cytogenetics, the technologies used to identify chromosomal rearrangements and copy number changes, and the applications for cytogenetics in biomedical diagnosis and research are presented and discussed.

  5. Exploring non-covalent interactions in guanine- and xanthine-based model DNA quadruplex structures: a comprehensive quantum chemical approach. (United States)

    Yurenko, Yevgen P; Novotný, Jan; Sklenář, Vladimir; Marek, Radek


    The study aimed to cast light on the structure and internal energetics of guanine- and xanthine-based model DNA quadruplexes and the physico-chemical nature of the non-covalent interactions involved. Several independent approaches were used for this purpose: DFT-D3 calculations, Quantum Theory of Atoms in Molecules, Natural Bond Orbital Analysis, Energy Decomposition Analysis, Compliance Constant Theory, and Non-Covalent Interaction Analysis. The results point to an excellent degree of structural and energetic compatibility between the two types of model quadruplexes. This fact stems from both the structural features (close values of van der Waals volumes, pore radii, geometrical parameters of the H-bonds) and the energetic characteristics (comparable values of the energies of formation). It was established that hydrogen bonding makes the greatest (∼50%) contribution to the internal stability of the DNA quadruplexes, whereas the aromatic base stacking and ion coordination terms are commensurable and account for the rest. Energy decomposition analysis performed for guanine (Gua) and xanthine (Xan) quartets B4 and higher-order structures consisting of two or three stacked quartets indicates that whereas Gua structures benefit from a high degree of H-bond cooperativity, Xan models are characterized by a more favorable and cooperative π-π stacking. The results of electron density topological analysis show that Na(+)/K(+) ion coordination deeply affects the network of non-covalent interactions in Gua models due to the change in the twist angle between the stacked tetrads. For Xan models, ion coordination makes tetrads in stacks more planar without changing the twist angle. Therefore, the presence of the ion seems to be essential for the formation of planar stacks in Xan-based DNA quadruplexes. Detailed study of the nature of ion-base coordination suggests that this interaction has a partially covalent character and cannot be considered as purely electrostatic

  6. A Theoretical Study of the Binding of [Re6Se8(OH2(H2O4] Rhenium Clusters to DNA Purine Base Guanine

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    Leonor Alvarado-Soto


    Full Text Available Hexanuclear rhenium complexes are promising candidates for use as antitumor drugs. However, to date, there has been no investigation into the nature of their binding to DNA. In this study, density functional theory (DFT was used to examine the binding of [Re6Se8(OH2(H2O4] to the DNA purine base guanine. The geometrical structures of cluster-guanine adducts in water were modeled at the zero order regular approximation (ZORA-PW91 level. Calculating the bond energies allowed us to compare the cis and trans forms of the cluster, and a possible manners of interaction between [Re6Se8(OH2(H2O3] clusters and DNA was obtained and explained.

  7. 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


    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 ...

  8. Voltammetric and electrochemical gravimetric selective detection of interactions between Tl(I) and guanine and the influence on activity of DNA drug-intercalators. (United States)

    Nowicka, Anna M; Mackiewicz, Marcin; Matysiak, Edyta; Krasnodebska-Ostrega, Beata; Stojek, Zbigniew


    The interactions of Tl(I), a well known toxic species, with selected oligonucleotides were examined. The oligonucleotide sequences selected for the investigation were taken from gene hOGG1 responsible for repairing of DNA damage. Cyclic voltammetry was particularly useful, since nitrogen N-7 in guanine can be electrooxidized while its binding with Tl(I) leads to the loss of electroactivity. So, this selected interaction could be quantitatively used in drawing Scatchard's plot and calculating the binding constants and the number of active sites in guanine molecules occupied by one metal ion. Further, we have shown that the presence of Tl(I) leads to a decrease in activity of doxorubicin (DOX), a popular anticancer drug, vs. DNA. The obtained circular dichroism (CD) spectra and the measurements with an electrochemical quartz crystal microbalance (EQCM) led to a conclusion that in the presence of monovalent thallium cations the DNA double helix was neither damaged/oxidized nor its conformation changed substantially.

  9. Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT in acute malaria

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    Woodberry Tonia


    Full Text Available Abstract Background The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. Methods PBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNγ ELISPOT assay and ELISA. Results HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-γ production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. Conclusion The prevalence of acute proliferative and convalescent IFNγ responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

  10. Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides. (United States)

    Karnawat, Vishakha; Gogia, Spriha; Balaram, Hemalatha; Puranik, Mrinalini


    Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non-covalent interactions stabilise the pre-transition state of the HGPRT-catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross-section in enzyme-bound 9-deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross-section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG-PRPP-HGPRT than in 9-deazahypoxanthine (9DAH)-PRPP-HGPRT.

  11. 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


    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.

  12. DPT tautomerisation of the wobble guanine·thymine DNA base mispair is not mutagenic: QM and QTAIM arguments. (United States)

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M


    We have shown for the first time, connecting QM methods with QTAIM analysis and using the methodology of the sweeps of the energetical, electron-topological and geometrical parameters, that the tautomerisation of the wobble guanine·thymine (wG·T) DNA base mispair into the wG(*)·T(*) base mispair induced by the double proton transfer (DPT), which undergoes a concerted asynchronous pathway, is not mutagenic. The wG·T → wG(*)·T(*) DPT tautomerisation does not result in the transition of the G base into its mutagenic tautomeric form G(*) able to mispair with the T base within the Watson-Crick base pairing scheme. This observation is explained by the so-called quantum protection of the wG·T DNA base mispair from its mutagenic tautomerisation - the dynamical non-stability of the tautomerised wG(*)·T(*) base mispair and significantly negative value of the Gibbs free energy of activation for the reverse reaction of the wG·T → wG(*)·T(*) DPT tautomerisation.

  13. Nucleotide excision repair in differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail:


    Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

  14. Empirical Bayes analysis of single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Ickstadt Katja


    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 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.

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

    Directory of Open Access Journals (Sweden)

    Simon Reed


    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.

  16. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions. (United States)

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao


    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation.

  17. 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


    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.

  18. (/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.


    (/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.

  19. Facilitation of ß-cell K(ATP) channel sulfonylurea sensitivity by a cAMP analog selective for the cAMP-regulated guanine nucleotide exchange factor Epac. (United States)

    Leech, Colin A; Dzhura, Igor; Chepurny, Oleg G; Schwede, Frank; Genieser, Hans-G; Holz, George G


    Clinical studies demonstrate that combined administration of sulfonylureas with exenatide can induce hypoglycemia in type 2 diabetic subjects. Whereas sulfonylureas inhibit ß-cell K(ATP) channels by binding to the sulfonylurea receptor-1 (SUR1), exenatide binds to the GLP-1 receptor, stimulates ß-cell cAMP production and activates both PKA and Epac. In this study, we hypothesized that the adverse in vivo interaction of sulfonylureas and exenatide to produce hypoglycemia might be explained by Epac-mediated facilitation of K(ATP) channel sulfonylurea sensitivity. We now report that the inhibitory action of a sulfonylurea (tolbutamide) at K(ATP) channels was facilitated by 2’-O-Me-cAMP, a selective activator of Epac. Thus, under conditions of excised patch recording, the dose-response relationship describing the inhibitory action of tolbutamide at human ß-cell or rat INS-1 cell K(ATP) channels was left-shifted in the presence of 2’-O-Me-cAMP, and this effect was abolished in INS-1 cells expressing a dominant-negative Epac2. Using an acetoxymethyl ester prodrug of an Epac-selective cAMP analog (8-pCP T-2’-O-Me-cAMP-AM), the synergistic interaction of an Epac activator and tolbutamide to depolarize INS-1 cells and to raise [Ca²(+)](i) was also measured. This effect of 8-pCP T-2’-O-Me-cAMP-AM correlated with its ability to stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis that might contribute to the changes in K(ATP) channel sulfonylurea-sensitivity reported here. On the basis of such findings, we propose that the adverse interaction of sulfonylureas and exenatide to induce hypoglycemia involves at least in part, a functional interaction of these two compounds to close K(ATP) channels, to depolarize ß-cells and to promote insulin secretion.

  20. Lithium, an inhibitor of cAMP-induced inositol 1,4,5-trisphosphate accumulation in Dictyostelium discoideum, inhibits activation of guanine-nucleotide-binding regulatory proteins, reduces activation of adenylylcyclase, but potentiates activation of guanyl

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Snaar-Jagalska, B. Ewa; Haastert, Peter J.M. van; Schaap, Pauline


    Li+ drastically alters pattern formation in Dictyostelium by inhibiting cAMP-induced prespore-gene expression and promoting cAMP-induced prestalk-gene expression. We reported previously that Li+ inhibits inositol monophosphatases in this organism and strongly reduces basal and cAMP-stimulated inosit

  1. Overview of the application of nucleotide in aquaculture

    Directory of Open Access Journals (Sweden)

    Hoang Do Huu


    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.

  2. Nucleotide Metabolism and its Control in Lactic Acid Bacteria

    DEFF Research Database (Denmark)

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


    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...... 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....

  3. Overview of the application of nucleotide in aquaculture

    Institute of Scientific and Technical Information of China (English)

    Hoang Do Huu


    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.

  4. Nucleotide excision repair of DNA: The very early history. (United States)

    Friedberg, Errol C


    This article, taken largely from the book Correcting the Blueprint of Life: An Historical Account of the Discovery of DNA Repair Mechanisms, summarizes the very early history of the discovery of nucleotide excision repair.

  5. Association study of nonsynonymous single nucleotide polymorphisms in schizophrenia

    DEFF Research Database (Denmark)

    Carrera, Noa; Arrojo, Manuel; Sanjuán, Julio


    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, ...

  6. The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Lykke, Anne; Rasmussen, Lene Juel


    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...... and DNA synthesis in part can be responsible for this link. This paper summarizes the parts of the metabolic pathways responsible for nucleotide metabolism that can be affected by mitochondrial dysfunction....

  7. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Handford M.


    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.

  9. Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation. (United States)

    Khalpey, Zain; Yuen, Ada H Y; Lavitrano, Marialuisa; McGregor, Christopher G A; Kalsi, Kameljit K; Yacoub, Magdi H; Smolenski, Ryszard T


    Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors.Ecto-5'-nucleotidase (E5'N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.

  10. Nuclear magnetic resonance studies of an N2-guanine adduct derived from the tumorigen dibenzo[a,l]pyrene in DNA: impact of adduct stereochemistry, size, and local DNA sequence on solution conformations. (United States)

    Rodríguez, Fabián A; Liu, Zhi; Lin, Chin H; Ding, Shuang; Cai, Yuqin; Kolbanovskiy, Alexander; Kolbanovskiy, Marina; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E


    The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide-DNA adducts formed include the stereoisomeric 14S and 14R trans-anti-DB[a,l]P-N(2)-dG and the stereochemically analogous 10S- and 10R-B[a]P-N(2)-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N(2)-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N(2)-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5'-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure-function relationship in NER.

  11. Phosphate-Modified Nucleotides for Monitoring Enzyme Activity. (United States)

    Ermert, Susanne; Marx, Andreas; Hacker, Stephan M


    Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

  12. Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB. (United States)

    Singh, Vipender; Fedeles, Bogdan I; Li, Deyu; Delaney, James C; Kozekov, Ivan D; Kozekova, Albena; Marnett, Lawrence J; Rizzo, Carmelo J; Essigmann, John M


    The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

  13. 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.

  14. Moss Phylogeny Reconstruction Using Nucleotide Pangenome of Complete Mitogenome Sequences. (United States)

    Goryunov, D V; Nagaev, B E; Nikolaev, M Yu; Alexeevski, A V; Troitsky, A V


    Stability of composition and sequence of genes was shown earlier in 13 mitochondrial genomes of mosses (Rensing, S. A., et al. (2008) Science, 319, 64-69). It is of interest to study the evolution of mitochondrial genomes not only at the gene level, but also on the level of nucleotide sequences. To do this, we have constructed a "nucleotide pangenome" for mitochondrial genomes of 24 moss species. The nucleotide pangenome is a set of aligned nucleotide sequences of orthologous genome fragments covering the totality of all genomes. The nucleotide pangenome was constructed using specially developed new software, NPG-explorer (NPGe). The stable part of the mitochondrial genome (232 stable blocks) is shown to be, on average, 45% of its length. In the joint alignment of stable blocks, 82% of positions are conserved. The phylogenetic tree constructed with the NPGe program is in good correlation with other phylogenetic reconstructions. With the NPGe program, 30 blocks have been identified with repeats no shorter than 50 bp. The maximal length of a block with repeats is 140 bp. Duplications in the mitochondrial genomes of mosses are rare. On average, the genome contains about 500 bp in large duplications. The total length of insertions and deletions was determined in each genome. The losses and gains of DNA regions are rather active in mitochondrial genomes of mosses, and such rearrangements presumably can be used as additional markers in the reconstruction of phylogeny.

  15. Monte carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. II. Comparisons of model predictions to measured data. (United States)

    Semenenko, V A; Stewart, R D


    Clustered damage sites other than double-strand breaks (DSBs) have the potential to contribute to deleterious effects of ionizing radiation, such as cell killing and mutagenesis. In the companion article (Semenenko et al., Radiat. Res. 164, 180-193, 2005), a general Monte Carlo framework to simulate key steps in the base and nucleotide excision repair of DNA damage other than DSBs is proposed. In this article, model predictions are compared to measured data for selected low-and high-LET radiations. The Monte Carlo model reproduces experimental observations for the formation of enzymatic DSBs in Escherichia coli and cells of two Chinese hamster cell lines (V79 and xrs5). Comparisons of model predictions with experimental values for low-LET radiation suggest that an inhibition of DNA backbone incision at the sites of base damage by opposing strand breaks is active over longer distances between the damaged base and the strand break in hamster cells (8 bp) compared to E. coli (3 bp). Model estimates for the induction of point mutations in the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene by ionizing radiation are of the same order of magnitude as the measured mutation frequencies. Trends in the mutation frequency for low- and high-LET radiation are predicted correctly by the model. The agreement between selected experimental data sets and simulation results provides some confidence in postulated mechanisms for excision repair of DNA damage other than DSBs and suggests that the proposed Monte Carlo scheme is useful for predicting repair outcomes.

  16. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.


    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  17. 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. (United States)

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


    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.

  18. Compositions and methods for detecting single nucleotide polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.


    Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

  19. Extracellular nucleotide derivatives protect cardiomyctes against hypoxic stress

    DEFF Research Database (Denmark)

    Golan, O; Issan, Y; Isak, A


    in cardioprotection against hypoxic stress has not been reported. OBJECTIVE: To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia. METHODS AND RESULTS: Rat cultured cardiomyocytes were treated with various extracellular nucleotides...... and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists...... (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y₂ -/- , P2Y₄ -/-, and P2Y₂ -/-/P2Y₄ -/- receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect...

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

    CERN Document Server

    Fialko, N S


    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.

  1. Effect of nucleotides on broiler performance and carcass yield

    Directory of Open Access Journals (Sweden)

    VC Pelícia


    Full Text Available This study aimed at evaluating the effect of nucleotides on the performance and carcass yield of broilers fed diets with no antibiotic growth promoters (AGP, anticoccidials, or animal feedstuffs. In the trial, 600 Ross 308 male broilers were distributed in a completely randomized experimental design into six treatments with four replicates of 25 birds each. Treatments consisted of a control diet (CD, CD + AGP, CD + 0.04%, CD + 0.05%, CD + 0.06%, and CD + 0.07% nucleotides. The experimental diets did not contain anticoccidials, and birds were vaccinated against coccidiosis at three days of age. No significant differences were detected among broilers submitted to the different treatments in none of the studied parameters. Under the conditions of this experiment, diets supplemented with nucleotides did not influence broiler performance or carcass yield at 42 days of age, and were not different from the feeds not containing any additive or with AGP.

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

    Directory of Open Access Journals (Sweden)

    Kevin H. Shaughnessy


    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.

  3. Nucleotide frequencies in human genome and fibonacci numbers. (United States)

    Yamagishi, Michel E Beleza; Shimabukuro, Alex Itiro


    This work presents a mathematical model that establishes an interesting connection between nucleotide frequencies in human single-stranded DNA and the famous Fibonacci's numbers. The model relies on two assumptions. First, Chargaff's second parity rule should be valid, and second, the nucleotide frequencies should approach limit values when the number of bases is sufficiently large. Under these two hypotheses, it is possible to predict the human nucleotide frequencies with accuracy. This result may be used as evidence to the Fibonacci string model that was proposed to the sequence growth of DNA repetitive sequences. It is noteworthy that the predicted values are solutions of an optimization problem, which is commonplace in many of nature's phenomena.

  4. Effect of nucleotides on broiler performance and carcass yield


    VC Pelícia; JR Sartori; KC Zavarize; AC Pezzato; AC Stradiotti; PC Araujo; MAO Mituo; LA Madeira


    This study aimed at evaluating the effect of nucleotides on the performance and carcass yield of broilers fed diets with no antibiotic growth promoters (AGP), anticoccidials, or animal feedstuffs. In the trial, 600 Ross 308 male broilers were distributed in a completely randomized experimental design into six treatments with four replicates of 25 birds each. Treatments consisted of a control diet (CD), CD + AGP, CD + 0.04%, CD + 0.05%, CD + 0.06%, and CD + 0.07% nucleotides. The experimental ...

  5. 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


    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.

  6. Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. (United States)

    Stengl, Bernhard; Reuter, Klaus; Klebe, Gerhard


    Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.

  7. Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase. (United States)

    Jin, Zhinan; Johnson, Kenneth A


    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.

  8. DNA Nucleotides Detection via capacitance properties of Graphene (United States)

    Khadempar, Nahid; Berahman, Masoud; Yazdanpanah, Arash


    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.

  9. A Laboratory Exercise for Genotyping Two Human Single Nucleotide Polymorphisms (United States)

    Fernando, James; Carlson, Bradley; LeBard, Timothy; McCarthy, Michael; Umali, Finianne; Ashton, Bryce; Rose, Ferrill F., Jr.


    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…

  10. DNA sequence representation by trianders and determinative degree of nucleotides

    Institute of Scientific and Technical Information of China (English)

    DUPLIJ Diana; DUPLIJ Steven


    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.

  11. Synthesis, bioanalysis and pharmacology of nucleoside and nucleotide analogs

    NARCIS (Netherlands)

    Jansen, R.S.


    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

  12. Environmental heat stress, hyperammonemia and nucleotide metabolism during intermittent exercise

    DEFF Research Database (Denmark)

    Mohr, Magni; Rasmussen, Peter; Drust, Barry


    Abstract  This study investigated the influence of environmental heat stress on ammonia (NH3) accumulation in relation to nucleotide metabolism and fatigue during intermittent exercise. Eight males performed 40 min of intermittent exercise (15 s at 306±22 W alternating with 15 s of unloaded cycli...

  13. The nucleotide sequences of two leghemoglobin genes from soybean

    DEFF Research Database (Denmark)

    Wiborg, O; Hyldig-Nielsen, J J; Jensen, E O


    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...

  14. Single nucleotide polymorphisms associated with rat expressed sequences

    NARCIS (Netherlands)

    Guryev, Victor; Berezikov, Eugene; Malik, Rainer; Plasterk, Ronald H A; Cuppen, Edwin


    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

  15. [Tabular excel editor for analysis of aligned nucleotide sequences]. (United States)

    Demkin, V V


    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.

  16. Single Nucleotide Polymorphisms Predict Symptom Severity of Autism Spectrum Disorder (United States)

    Jiao, Yun; Chen, Rong; Ke, Xiaoyan; Cheng, Lu; Chu, Kangkang; Lu, Zuhong; Herskovits, Edward H.


    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)…

  17. Nucleotide excision repair: ERCC1 and TFIIH complexes

    NARCIS (Netherlands)

    A.J. van Vuuren (Hanneke)


    textabstractDNA is the carrier of genetic information in living organisms. The information stored in the nucleotide sequence of DNA is transmitted to the offspring by generating identical copies of the parental DNA molecules. Damage in DNA can cause loss of genetic information. Nevertheless, the DNA

  18. Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis (United States)


    Montie, S. Kadis, and S. I. Ajl (ed.), Microbial toxins, vol. 3. Academic Press, Inc., New York. 23. Little, S. F., and G. B. Knudaon. 1986...Takkinen, and L. Kaariainen. 1981. Nucleotide sequence of the promoter and NHa-terminal signal peptide region of the a- amylase gene from Bacillus

  19. 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.


    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

  20. 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.

  1. Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment. (United States)

    Gopalakrishnan, Jayachandran; Chim, Yiu-Cheung Frederick; Ha, Andrew; Basiri, Marcus L; Lerit, Dorothy A; Rusan, Nasser M; Avidor-Reiss, Tomer


    Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM). PCM assembly is a tightly regulated, critical step that determines the size and capability of centrosomes. Here, we report a role for tubulin in regulating PCM recruitment through the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These results suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, the regulation of PCM recruitment by tubulin depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules. On the basis of its guanine-bound state, tubulin can act as a molecular switch in PCM recruitment.

  2. Computational Studies of the Binding of Ligands to the Guanine Riboswitch Aptamer%鸟嘌呤核糖开关识别配体小分子

    Institute of Scientific and Technical Information of China (English)

    马爱静; 扈国栋; 王吉华


    The Guanine Riboswitch is genetic regulatory element found in the non translation region loca‐ted of mRNA .It can be directly combined with organic metabolites to induces conformational changes to control gene expression .Recognition of them has highly specificity and selectivity .In this work ,we stud‐ied the nature of the Guanine Riboswitch and ligands by molecular dynamics simulation .Then ,the calcu‐lation of the relative binding free energy of them by Thermodynamic Integration .The study found that the binding pocket of Riboswitch form Waston -Crick base pairs with ligand and we learned that the affinities are different when the binding of different ligands to Riboswitch .These data indicate a more complicated relationship between the 6-position of the ligand and the RNA and The 2-position of the purine ligand is essential for the recognition .As an ideal target of new drugs ,the Guanine Riboswitch has an important role in the design and development of new drugs .%鸟嘌呤核糖开关是一类位于m RN A 的非翻译区,可直接结合有机代谢物,通过调整自身构象变化从而调控基因表达的m RN A元件。鸟嘌呤核糖开关识别配体具有高度特异性和选择性。用动力学模拟的方法,研究鸟嘌呤核糖开关特异识别配体小分子的本质和用热力学积分算法计算核糖开关与配体分子的相对结合自由能。研究发现,核糖开关的结合口袋与配体通过氢键形式相互作用,不同配体与核糖开关结合的亲和力不同。嘌呤配体2-号位对核糖开关的结合必不可少,6-号位对核糖开关的结合关系复杂。

  3. 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


    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.

  4. Identification of the Structural Features of Guanine Derivatives as MGMT Inhibitors Using 3D-QSAR Modeling Combined with Molecular Docking. (United States)

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


    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.

  5. Pseudoscorpion mitochondria show rearranged genes and genome-wide reductions of RNA gene sizes and inferred structures, yet typical nucleotide composition bias

    Directory of Open Access Journals (Sweden)

    Ovchinnikov Sergey


    Full Text Available Abstract Background Pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. No mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of Chelicerata possess unusual features, including short rRNA genes and tRNA genes that lack sequence to encode arms of the canonical cloverleaf-shaped tRNA. Additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the major coding strand of their mitochondrial genomes. Results We sequenced the mitochondrial genomes of two divergent taxa from the chelicerate order Pseudoscorpiones. We find that these genomes possess unusually short tRNA genes that do not encode cloverleaf-shaped tRNA structures. Indeed, in one genome, all 22 tRNA genes lack sequence to encode canonical cloverleaf structures. We also find that the large ribosomal RNA genes are substantially shorter than those of most arthropods. We inferred secondary structures of the LSU rRNAs from both pseudoscorpions, and find that they have lost multiple helices. Based on comparisons with the crystal structure of the bacterial ribosome, two of these helices were likely contact points with tRNA T-arms or D-arms as they pass through the ribosome during protein synthesis. The mitochondrial gene arrangements of both pseudoscorpions differ from the ancestral chelicerate gene arrangement. One genome is rearranged with respect to the location of protein-coding genes, the small rRNA gene, and at least 8 tRNA genes. The other genome contains 6 tRNA genes in novel locations. Most chelicerates with rearranged mitochondrial genes show a genome-wide reversal of the CA nucleotide bias typical for arthropods on their major coding strand, and instead possess a GT bias. Yet despite their extensive rearrangement, these pseudoscorpion mitochondrial genomes possess a CA bias on the major coding strand. Phylogenetic

  6. From lin-Benzoguanines to lin-Benzohypoxanthines as Ligands for Zymomonas mobilis tRNA-Guanine Transglycosylase: Replacement of Protein-Ligand Hydrogen Bonding by Importing Water Clusters.

    NARCIS (Netherlands)

    Barandun, L.J.; Immekus, F.; Kohler, P.C.; Tonazzi, S.; Wagner, B.; Wendelspiess, S.; Ritschel, T.; Heine, A.; Kansy, M.; Klebe, G.; Diederich, F.


    The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of Shig

  7. Exonuclease activity and P nucleotide addition in the generation of the expressed immunoglobulin repertoire

    Directory of Open Access Journals (Sweden)

    Sewell William


    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.

  8. The electronic structure of the four nucleotide bases in DNA, of their stacks, and of their homopolynucleotides in the absence and presence of water (United States)

    Ladik, János; Bende, Attila; Bogár, Ferenc


    Using the ab initio Hartree-Fock crystal orbital method in its linear combination of atomic orbital form, the energy band structure of the four homo-DNA-base stacks and those of poly(adenilic acid), polythymidine, and polycytidine were calculated both in the absence and presence of their surrounding water molecules. For these computations Clementi's double ζ basis set was applied. To facilitate the interpretation of the results, the calculations were supplemented by the calculations of the six narrow bands above the conduction band of poly(guanilic acid) with water. Further, the sugar-phosphate chain as well as the water structures around poly(adenilic acid) and polythymidine, respectively, were computed. Three important features have emerged from these calculations. (1) The nonbase-type or water-type bands in the fundamental gap are all close to the corresponding conduction bands. (2) The very broad conduction band (1.70eV) of the guanine stack is split off to seven narrow bands in the case of poly(guanilic acid) (both without and with water) showing that in the energy range of the originally guanine-stack-type conduction band, states belonging to the sugar, to PO4-, to Na+, and to water mix with the guanine-type states. (3) It is apparent that at the homopolynucleotides with water in three cases the valence bands are very similar (polycytidine, because it has a very narrow valence band, does not fall into this category). We have supplemented these calculations by the computation of correlation effects on the band structures of the base stacks by solving the inverse Dyson equation in its diagonal approximation taken for the self-energy the MP2 many body perturbation theory expression. In all cases the too large fundamental gap decreased by 2-3eV. In most cases the widths of the valence and conduction bands, respectively, decreased (but not in all cases). This unusual behavior is most probably due to the rather large complexity of the systems. From all this

  9. Theoretical calculation of the NMR spin-spin coupling constants and the NMR shifts allow distinguishability between the specific direct and the water-mediated binding of a divalent metal cation to guanine. (United States)

    Sychrovský, Vladimír; Sponer, Jirí; Hobza, Pavel


    The calculated intermolecular and intramolecular indirect NMR spin-spin coupling constants and NMR shifts were used for the discrimination between the inner-shell and the outer-shell binding motif of hydrated divalent cations Mg(2+) or Zn(2+) with a guanine base. The intermolecular coupling constants (1)J(X,O6) and (1)J(X,N7) (X = Mg(2+), Zn(2+)) can be unambiguously assigned to the specific inner-shell binding motif of the hydrated cation either with oxygen O6 or with nitrogen N7 of guanine. The calculated coupling constants (1)J(Mg,O6) and (1)J(Zn,O6) were 6.2 and -17.5 Hz, respectively, for the inner-shell complex of cation directly interacting with oxygen O6 of guanine. For the inner-shell coordination of the cation at nitrogen N7, the calculated coupling constants (1)J(Mg,N7) and (1)J(Zn,N7) were 5.6 and -36.5 Hz, respectively. When the binding of the cation is water-mediated, the coupling constant is zero. To obtain reliable shifts in NMR parameters, hydrated guanine was utilized as the reference state. The calculated change of NMR spin-spin coupling constants due to the hydration and coordination of the cation with guanine is caused mainly by the variation of Fermi-contact coupling contribution while the variation of diamagnetic spin-orbit, paramagnetic spin-orbit, and spin-dipolar coupling contributions is small. The change of s-character of guanine sigma bonding, sigma antibonding, and lone pair orbitals upon the hydration and cation coordination (calculated using the Natural Bond Orbital analysis) correlates with the variation of the Fermi-contact term. The calculated NMR shifts delta(N7) of -15.3 and -12.2 ppm upon the coordination of Mg(2+) and Zn(2+) ion are similar to the NMR shift of 19.6 ppm toward the high field measured by Tanaka for N7 of guanine upon the coordination of the Cd(2+) cation (Tanaka, Y.; Kojima, C.; Morita, E. H.; Kasai. Y.; Yamasaki, K.; Ono, A.; Kainosho, M.; Taira, K. J. Am. Chem. Soc. 2002, 124, 4595-4601). The present data

  10. 氮掺杂石墨烯修饰电极的制备及对鸟嘌呤的电催化氧化%Fabrication of N-doped Graphene-modified Electrode and Its Electrocatalytic Oxidation of Guanine

    Institute of Scientific and Technical Information of China (English)



    N-doped graphene (NG) was synthesized by thermal annealing at high temperature. The electrocatalytic oxidation of guanine at the NG-modified glass carbon electrode(GCE) was investigated. The effects of pH value, scanning rate and guanine concentration were discussed. The results showed that the oxidation process of guanine at the NG/GCE was an irreversible process and the adsorption of guanine at the modified electrode surface was increased. The modified electrode had an obvious electrocatalytic activity on guanine and decreased the oxidation potential of guanine. In a phosphate buffer solution(pH = 7. 0),the oxidation peak current of guanine and its concentration had a linear relationship in a range of 5. OX 1CTS-1. OX 10~1mol/L at the modified electrode and the detection limit was of 1.0X10~6mol/L.%采用高温热退火方法制备了氮掺杂的石墨烯,并制备了氮掺杂石墨烯修饰玻碳电极(NG/GCE),研究其对鸟嘌呤的电催化氧化作用.实验考察了溶液pH值、扫速、鸟嘌呤浓度的影响.结果表明,鸟嘌呤在NG/GCE上的氧化是不可逆过程,修饰电极可以增强鸟嘌呤在电极表面的吸附,对鸟嘌呤具有很好的电催化氧化性能,降低了鸟嘌呤氧化电位.在pH=7.0的磷酸盐缓冲溶液中检测鸟嘌呤,其氧化峰电流在5.0×10-6~1.0×10-4 mol/L浓度范围内呈良好的线性关系,检出限(3σ)为1.0×10-6 mol/L.

  11. The nucleotide exchange factors of Hsp70 molecular chaperone

    Directory of Open Access Journals (Sweden)

    Andreas eBracher


    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.

  12. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.


    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.

  13. Genome-wide patterns of nucleotide polymorphism in domesticated rice

    DEFF Research Database (Denmark)

    Caicedo, Ana L; Williamson, Scott H; Hernandez, Ryan D


    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...... 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...

  14. N,N,O and N,O,N Meridional cis Coordination of Two Guanines to Copper(II) by d(CGCGCG)2. (United States)

    Rohner, Melanie; Medina-Molner, Alfredo; Spingler, Bernhard


    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.

  15. 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 File URL: File size: 19 MB File name: FASTA: File URL: About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Nucleotide Sequence - KOME | LSDB Archive ...

  16. Adenine nucleotide concentrations in patients with erythrocyte autoantibodies.


    Strong, V F; Sokol, R J; Rodgers, S A; Hewitt, S.


    Erythrocyte adenine nucleotide concentrations were measured in 154 patients with erythrocyte autoantibodies and 811 normal subjects using a luciferin-luciferase bioluminescent assay. The patients were initially divided into haemolysing and non-haemolysing groups. Red cell adenosine triphosphate (ATP) concentrations were significantly raised in the 96 patients with active haemolysis compared with the normal subjects and with the 58 patients in the non-haemolysing group. Although the patients c...

  17. Flavin nucleotides in human lens: regional distribution in brunescent cataracts. (United States)

    Bhat, K S; Nayak, S


    The biochemical mechanism(s) underlying brunescent cataracts remain unclear. Oxidative stress due to reactive oxygen species may have a role in the pigmentation process in eye lens. We have analysed human cataractous lenses for flavins by high-performance liquid chromatography (HPLC), since flavins are light sensitive and act as endogenous sensitizers generating reactive oxygen species in the eye. The most significant observation in this study is that higher levels of flavin nucleotides occur in brown lens compared to yellow lens. The concentration of flavin nucleotides (flavin monouncleotide, FMN + flavin adenine dinucleotide, FAD) was highest in the nuclear region of the lens followed by the cortical and capsule-epithelial regions. However, the ratio of FAD/FMN was lowest in the nuclear region of the lens followed by other regions. On the other hand, riboflavin was not detected in any of the lens (cataractous) regions. These results suggest that the observed increase in flavin nucleotides in the ocular tissue could contribute towards deepening of lens pigmentation.

  18. Nucleotide Sequencing and Identification of Some Wild Mushrooms

    Directory of Open Access Journals (Sweden)

    Sudip Kumar Das


    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.

  19. Genome-wide patterns of nucleotide polymorphism in domesticated rice.

    Directory of Open Access Journals (Sweden)

    Ana L Caicedo


    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.

  20. Nucleotide sequencing and identification of some wild mushrooms. (United States)

    Das, Sudip Kumar; Mandal, Aninda; Datta, Animesh K; Gupta, Sudha; Paul, Rita; Saha, Aditi; Sengupta, Sonali; Dubey, Priyanka Kumari


    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.

  1. Single nucleotide polymorphisms and linkage disequilibrium in sunflower. (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


    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 ( = 0.0094) than wild populations ( = 0.0128). Mean haplotype diversity was 0.74. When extraploted across the genome ( approximately 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.

  2. Spontaneous formation and base pairing of plausible prebiotic nucleotides in water. (United States)

    Cafferty, Brian J; Fialho, David M; Khanam, Jaheda; Krishnamurthy, Ramanarayanan; Hud, Nicholas V


    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.

  3. 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: [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)


    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.

  4. n-Nucleotide circular codes in graph theory. (United States)

    Fimmel, Elena; Michel, Christian J; Strüngmann, Lutz


    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

  5. The nucleotide sequence and genome organization of Plasmopara halstedii virus

    Directory of Open Access Journals (Sweden)

    Göpfert Jens C


    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

  6. Performance-enhancing effects of dietary nucleotides: do mitochondria play a role?

    Directory of Open Access Journals (Sweden)

    Sergej M. Ostojic


    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.

  7. Effects of bovine cytochrome P450 single-nucleotide polymorphism, forage type and body condition on production traits in cattle. (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


    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.

  8. Guanine polynucleotides are self-antigens for human natural autoantibodies and are significantly reduced in the human genome. (United States)

    Fattal, Ittai; Shental, Noam; Ben-Dor, Shifra; Molad, Yair; Gabrielli, Armando; Pokroy-Shapira, Elisheva; Oren, Shirly; Livneh, Avi; Langevitz, Pnina; Zandman-Goddard, Gisele; Sarig, Ofer; Margalit, Raanan; Gafter, Uzi; Domany, Eytan; Cohen, Irun R


    In the course of investigating anti-DNA autoantibodies, we examined IgM and IgG antibodies to poly-G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20-mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo-nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti-G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170,000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain-associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti-G20 antibodies; so natural anti-G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti-G20 antibodies in human health and disease and of runs of G20 in the human genome.

  9. 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


    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...

  10. Nucleotide sequences specific to Brucella and methods for the detection of Brucella (United States)

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.


    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.

  11. Bioinformatics comparison of sulfate-reducing metabolism nucleotide sequences (United States)

    Tremberger, G.; Dehipawala, Sunil; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.


    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.

  12. Pinched flow fractionation devices for detection of single nucleotide polymorphisms

    DEFF Research Database (Denmark)

    Larsen, Asger Vig; Poulsen, Lena; Birgens, Henrik


    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...... with 13 mu m deep channels were fabricated by thermal nanoimprint lithography ( NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 mu m...

  13. Patterns of nucleotides that flank substitutions in human orthologous genes

    Directory of Open Access Journals (Sweden)

    Huang Zhuoran


    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

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

    DEFF Research Database (Denmark)

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


    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...... the sensor bias current to magnetize magnetic beads in the vicinity of the sensor. The method allows for real-time measurements of the specific bead binding to the sensor surface during DNA hybridization and washing. Compared to other magnetic biosensing platforms, our approach eliminates the need...... for external electromagnets and thus allows for miniaturization of the sensor platform....

  15. The complete nucleotide sequence of pelargonium leaf curl virus. (United States)

    McGavin, Wendy J; MacFarlane, Stuart A


    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.

  16. Copper intoxication inhibits aerobic nucleotide synthesis in Streptococcus pneumoniae (United States)

    Johnson, Michael D. L.; Kehl-Fie, Thomas E.; Rosch, Jason W.


    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

  17. Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures. (United States)

    Abi, Alireza; Ferapontova, Elena E


    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.

  18. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry. (United States)

    Xiao, Yongsheng; Wang, Yinsheng


    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.

  19. Dietary nucleotides influence immune responses and intestinal morphology of red drum Sciaenops ocellatus. (United States)

    Cheng, Zhenyan; Buentello, Alejandro; Gatlin, Delbert M


    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.

  20. Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams (United States)

    Shimamura, Shigeru; Kaneko, Takashi; Ozawa, Genki; Matsumoto, Mamiko Nishino; Koshiishi, Takeru; Takaki, Yoshihiro; Kato, Chiaki; Takai, Ken; Yoshida, Takao; Fujikura, Katsunori; Barry, James P.


    Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; uvrA, uvrB, uvrC, uvrD, uvrD paralog [uvrDp] and mfd) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While uvrA, uvrDp and mfd were conserved in all symbionts, uvrB and uvrC were degraded in all clade I symbionts but were apparently intact in clade II symbionts. UvrD was disrupted in two clade II symbionts. Among the intact genes in Ca. Vesicomyosocius okutanii (clade I), expressions of uvrD and mfd were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of uvrA and uvrDp were not. In contrast, all intact genes were expressed in the symbiont of Calyptogena pacifica (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from Bathymodiolus septemdierum were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and mutY in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome. PMID:28199404

  1. Solid-state NMR [13C,15N] resonance assignments of the nucleotide-Binding Domain of a bacterial Cyclic Nucleotide-Gated Channel

    NARCIS (Netherlands)

    Cukkemane, A.A.; Nand, D.; Gradmann, S.H.E.; Weingarth, M.H.; Kaupp, U.B.; Baldus, M.


    Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-sta

  2. Bayesian selection of nucleotide substitution models and their site assignments. (United States)

    Wu, Chieh-Hsi; Suchard, Marc A; Drummond, Alexei J


    Probabilistic inference of a phylogenetic tree from molecular sequence data is predicated on a substitution model describing the relative rates of change between character states along the tree for each site in the multiple sequence alignment. Commonly, one assumes that the substitution model is homogeneous across sites within large partitions of the alignment, assigns these partitions a priori, and then fixes their underlying substitution model to the best-fitting model from a hierarchy of named models. Here, we introduce an automatic model selection and model averaging approach within a Bayesian framework that simultaneously estimates the number of partitions, the assignment of sites to partitions, the substitution model for each partition, and the uncertainty in these selections. This new approach is implemented as an add-on to the BEAST 2 software platform. We find that this approach dramatically improves the fit of the nucleotide substitution model compared with existing approaches, and we show, using a number of example data sets, that as many as nine partitions are required to explain the heterogeneity in nucleotide substitution process across sites in a single gene analysis. In some instances, this improved modeling of the substitution process can have a measurable effect on downstream inference, including the estimated phylogeny, relative divergence times, and effective population size histories.

  3. Identification of novel cyclic nucleotide binding proteins in Trypanosoma cruzi. (United States)

    Jäger, Adriana V; De Gaudenzi, Javier G; Mild, Jesica G; Mc Cormack, Bárbara; Pantano, Sergio; Altschuler, Daniel L; Edreira, Martin M


    Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasite's life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known about cAMP's mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A (PKA) has been cloned and characterised in this parasite; however experimental evidence indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new cAMP binding proteins as potential cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a combination of search methods 27 proteins with putative cNMP binding domains (CBDs) were identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, with sequences segregated into two main branches: one containing kinases-like proteins and the other gathering hypothetical proteins with different function or no other known. Comparative modelling of the strongest candidates provides support for the hypothesis that these proteins may give rise to structurally viable cyclic nucleotide binding domains. Pull-down and nucleotide displacement assays strongly suggest that TcCLB.508523.80 could bind cAMP and eventually be a new putative PKA-independent cAMP effector in T. cruzi.

  4. Structure and function of nucleotide sugar transporters: Current progress

    Directory of Open Access Journals (Sweden)

    Barbara Hadley


    Full Text Available The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST. Therefore in this review we will provide the current state-of-play with respect to the structure–function relationship of the (CST. In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

  5. Detecting Single-Nucleotide Substitutions Induced by Genome Editing. (United States)

    Miyaoka, Yuichiro; Chan, Amanda H; Conklin, Bruce R


    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.

  6. Allosteric interactions of DNA and nucleotides with S. cerevisiae RSC. (United States)

    Malik, Shuja Shafi; Rich, Evan; Viswanathan, Ramya; Cairns, Bradley R; Fischer, Christopher J


    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.

  7. Single Nucleotide Polymorphism Analysis of Protamine Genes in Infertile Men

    Directory of Open Access Journals (Sweden)

    Ahamad Salamian


    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.

  8. Extracellular ATP and other nucleotides-ubiquitous triggers of intercellular messenger release. (United States)

    Zimmermann, Herbert


    Extracellular nucleotides, and ATP in particular, are cellular signal substances involved in the control of numerous (patho)physiological mechanisms. They provoke nucleotide receptor-mediated mechanisms in select target cells. But nucleotides can considerably expand their range of action. They function as primary messengers in intercellular communication by stimulating the release of other extracellular messenger substances. These in turn activate additional cellular mechanisms through their own receptors. While this applies also to other extracellular messengers, its omnipresence in the vertebrate organism is an outstanding feature of nucleotide signaling. Intercellular messenger substances released by nucleotides include neurotransmitters, hormones, growth factors, a considerable variety of other proteins including enzymes, numerous cytokines, lipid mediators, nitric oxide, and reactive oxygen species. Moreover, nucleotides activate or co-activate growth factor receptors. In the case of hormone release, the initially paracrine or autocrine nucleotide-mediated signal spreads through to the entire organism. The examples highlighted in this commentary suggest that acting as ubiquitous triggers of intercellular messenger release is one of the major functional roles of extracellular nucleotides. While initiation of messenger release by nucleotides has been unraveled in many contexts, it may have been overlooked in others. It can be anticipated that additional nucleotide-driven messenger functions will be uncovered with relevance for both understanding physiology and development of therapy.

  9. 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:


    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.

  10. MS-CASPT2 study of hole transfer in guanine-indole complexes using the generalized Mulliken-Hush method: effective two-state treatment. (United States)

    Butchosa, C; Simon, S; Blancafort, L; Voityuk, A


    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.

  11. Computational learning on specificity-determining residue-nucleotide interactions

    KAUST Repository

    Wong, Ka-Chun


    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.

  12. iCLIP: protein-RNA interactions at nucleotide resolution. (United States)

    Huppertz, Ina; Attig, Jan; D'Ambrogio, Andrea; Easton, Laura E; Sibley, Christopher R; Sugimoto, Yoichiro; Tajnik, Mojca; König, Julian; Ule, Jernej


    RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.

  13. Milestones in the discovery of antiviral agents: nucleosides and nucleotides

    Directory of Open Access Journals (Sweden)

    Erik de Clercq


    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.

  14. Computational identification of candidate nucleotide cyclases in higher plants

    KAUST Repository

    Wong, Aloysius Tze


    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.

  15. Large nucleotide-dependent conformational change in Rab28

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sung Haeng; Baek, Kyuwon; Dominguez, Roberto (UPENN-MED)


    Rab GTPases are essential regulators of membrane trafficking. We report crystal structures of Rab28 in the active (GppNHp-bound) and inactive (GDP-3'P-bound) forms at 1.5 and 1.1 {angstrom} resolution. Rab28 is a distant member of the Rab family. While the overall fold of Rab28 resembles that of other Rab GTPases, it undergoes a larger nucleotide-dependent conformational change than other members of this family. Added flexibility resulting from a double-glycine motif at the beginning of switch 2 might partially account for this observation. The double-glycine motif, which is conserved in the Arf family, only occurs in Rab28 and Rab7B of the Rab family, and may have a profound effect on their catalytic activities.

  16. Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae. (United States)

    Guintini, Laetitia; Charton, Romain; Peyresaubes, François; Thoma, Fritz; Conconi, Antonio


    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.

  17. Efficient fidelity control by stepwise nucleotide selection in polymerase elongation

    CERN Document Server

    Yu, Jin


    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...

  18. A nucleotide-level coarse-grained model of RNA

    CERN Document Server

    Šulc, Petr; Ouldridge, Thomas E; Doye, Jonathan P K; Louis, Ard A


    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.

  19. 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


    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.

  20. 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.

  1. ENGINES: exploring single nucleotide variation in entire human genomes

    Directory of Open Access Journals (Sweden)

    Salas Antonio


    Full Text Available Abstract Background Next generation ultra-sequencing technologies are starting to produce extensive quantities of data from entire human genome or exome sequences, and therefore new software is needed to present and analyse this vast amount of information. The 1000 Genomes project has recently released raw data for 629 complete genomes representing several human populations through their Phase I interim analysis and, although there are certain public tools available that allow exploration of these genomes, to date there is no tool that permits comprehensive population analysis of the variation catalogued by such data. Description We have developed a genetic variant site explorer able to retrieve data for Single Nucleotide Variation (SNVs, population by population, from entire genomes without compromising future scalability and agility. ENGINES (ENtire Genome INterface for Exploring SNVs uses data from the 1000 Genomes Phase I to demonstrate its capacity to handle large amounts of genetic variation (>7.3 billion genotypes and 28 million SNVs, as well as deriving summary statistics of interest for medical and population genetics applications. The whole dataset is pre-processed and summarized into a data mart accessible through a web interface. The query system allows the combination and comparison of each available population sample, while searching by rs-number list, chromosome region, or genes of interest. Frequency and FST filters are available to further refine queries, while results can be visually compared with other large-scale Single Nucleotide Polymorphism (SNP repositories such as HapMap or Perlegen. Conclusions ENGINES is capable of accessing large-scale variation data repositories in a fast and comprehensive manner. It allows quick browsing of whole genome variation, while providing statistical information for each variant site such as allele frequency, heterozygosity or FST values for genetic differentiation. Access to the data mart

  2. Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection. (United States)

    Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun


    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.

  3. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms. (United States)

    Matsuda, Kazuyuki; Honda, Takayuki


    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).

  4. 77 FR 65537 - Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence... (United States)


    ... 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...

  5. Cyclic nucleotides and radioresistance. Report 2. Effect of cyclic nucleotides on mammalian radioresistance and tissular oxygen tension. [X rays; mice

    Energy Technology Data Exchange (ETDEWEB)

    Kulinskii, V.I.


    In experiments on mice, 3',5'-AMP, N/sup 6/,O/sup 2/-dibutyryl-3',5'-AMP (DB), 2',3'-AMP, theophylline, papaverine, imidazole, and nicotinate presented neither a sensitizing or radioprotective (RPE) effect. However, preadministration of ..beta..-adrenolytic agents revealed a moderate RPE in DB. Probably, along with the RPE, there is an antiprotective component in the action of cyclic nucleotides (CN), which is expressed via ..beta..-receptors. The absence of RPE in 3',5'-AMP, 2',3'-AMP, DB, theophylline, and phentolamine is combined with decline of tissular pO/sub 2/. The significance of this phenomenon is discussed.

  6. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa). (United States)

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P


    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 s. 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.

  7. 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


    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.

  8. Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA. (United States)

    Catalano, Michael J; Liu, Shuo; Andersen, Nisana; Yang, Zhiyu; Johnson, Kevin M; Price, Nathan E; Wang, Yinsheng; Gates, Kent S


    A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

  9. Cytosine-Phosphorothionate-Guanine Oligodeoxynucleotides Exacerbates Hemophagocytosis by Inducing Tumor Necrosis Factor-Alpha Production in Mice after Bone Marrow Transplantation. (United States)

    Liu, Jiajia; Guo, Yong-Mei; Onai, Nobuyuki; Ohyagi, Hideaki; Hirokawa, Makoto; Takahashi, Naoto; Tagawa, Hiroyuki; Ubukawa, Kumi; Kobayashi, Isuzu; Tezuka, Hiroyuki; Minamiya, Yoshihiro; Ohteki, Toshiaki; Sawada, Kenichi


    Hemophagocytic syndrome (HPS) is frequently associated with hematopoietic stem cell transplantation and is treated with some benefit derived from TNF-α inhibitors. However, the mechanisms of how HPS occurs and how a TNF-α inhibitor exerts some benefit to HPS management have remained unclear. We evaluated the effect of toll-like receptor (TLR) ligands, especially focusing on cytosine-phosphorothionate-guanine oligodeoxynucleotide (CpG), a TLR9 ligand, on HPS in mice that underwent transplantation with syngeneic or allogeneic bone marrow (BM) cells (Syn-BMT, Allo-BMT), or with allogeneic BM cells plus splenocytes to promote graft-versus-host disease (GVHD mice). Hemophagocytosis was a common feature early after all BMT, but it subsided in Syn-BMT and Allo-BMT mice. In GVHD mice, however, hemophagocytosis persisted and was accompanied by upregulated production of IFN-γ but not TNF-α, and it was suppressed by blockade of IFN-γ but not TNF-α. A single injection of the TLR9 ligand CpG promoted HPS in all BMT mice and was lethal in GVHD mice, accompanied by greatly upregulated production of TNF-α, IL-6, and IFN-γ. Blocking of TNF-α, but not IL-6 or IFN-γ, suppressed CpG-induced HPS in all BMT mice and rescued GVHD mice from CpG-induced mortality. Thus, TLR9 signaling mediates TNF-α-driven HPS in BMT mice and is effectively treated through TNF-α inhibition.

  10. Direct detection of single-nucleotide polymorphisms in bacterial DNA by SNPtrap

    DEFF Research Database (Denmark)

    Grønlund, Hugo Ahlm; Moen, Birgitte; Hoorfar, Jeffrey


    A major challenge with single-nucleotide polymorphism (SNP) fingerprinting of bacteria and higher organisms is the combination of genome-wide screenings with the potential of multiplexing and accurate SNP detection. Single-nucleotide extension by the minisequencing principle represents a technolo...

  11. Rationally designed squaryldiamides - a novel class of sugar-nucleotide mimics? (United States)

    Niewiadomski, Sven; Beebeejaun, Zeenat; Denton, Helen; Smith, Terry K; Morris, Richard J; Wagner, Gerd K


    Sugar-nucleotides such as GDP-mannose, GDP-fucose and UDP-glucose are important biomolecules with a central role in carbohydrate and glycoconjugate biosynthesis, metabolism and cell signalling. Analogues and mimics of naturally occurring sugar-nucleotides are sought after as chemical tools and inhibitor candidates for sugar-nucleotide-dependent enzymes including glycosyltransferases. Many sugar-nucleotides bind to their target glycosyltransferases via coordination of the diphosphate group to a divalent metal cofactor in the active site. The identification of uncharged, chemically stable surrogates for the diphosphate group, with the ability to coordinate to a divalent metal, is therefore an important design criteria for the development of sugar-nucleotide mimics. Here, we describe the rational design and synthesis of a novel class of sugar-nucleotide mimics based on a squaryldiamide scaffold, an uncharged phosphate isostere. We demonstrate by comprehensive NMR titration experiments that the new sugar-nucleotide mimics coordinate efficiently to Mg(2+), and provide results from biological studies with a therapeutically relevant mannosyltransferase from Trypanosoma brucei. Our findings suggest that squaryldiamides are a promising template for the development of sugar-nucleotide mimics, and illustrate the considerable potential of the squarylamide group as a fragment for inhibitor design.

  12. Energy efficiency trade-offs drive nucleotide usage in transcribed regions. (United States)

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J


    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.

  13. ADH single nucleotide polymorphism associations with alcohol metabolism in vivo (United States)

    Birley, Andrew J.; James, Michael R.; Dickson, Peter A.; Montgomery, Grant W.; Heath, Andrew C.; Martin, Nicholas G.; Whitfield, John B.


    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

  14. Identifying sigma70 promoters with novel pseudo nucleotide composition. (United States)

    Lin, Hao; Liang, Zhi-Yong; Tang, Hua; Chen, Wei


    Promoters are DNA regulatory elements located directly upstream or at the 5' end of the transcription initiation site (TSS), which are in charge of gene transcription initiation. With the completion of a large number of microorganism genomics, it is urgent to predict promoters accurately in bacteria by using computational method. In this work, a sequence-based predictor named "iPro70-PseZNC" was designed for identifying sigma70 promoters in prokaryote. In the predictor, the samples of DNA sequences are formulated by a novel pseudo nucleotide composition, called PseZNC, into which the multi-window Z-curve composition and six local DNA structural properties are incorporated. In the 5-fold cross-validation, the area under the curve of receiver operating characteristic of 0.909 was obtained on our benchmark dataset, indicating that the proposed predictor is promising and will provide important guide in this area. Further studies showed that the performance of PseZNC is better than it of multi-window Z-curve composition. For the sake of convenience for researchers, a user-friendly online service was established and can be freely accessible at The PseZNC approach can be also extended to other DNA-related problems.

  15. Species radiation by DNA replication that systematically exchanges nucleotides? (United States)

    Seligmann, Hervé


    RNA and DNA syntheses share many properties. Therefore, the existence of 'swinger' RNAs, presumed 'orphan' transcripts matching genomic sequences only if transcription systematically exchanged nucleotides, suggests replication producing swinger DNA. Transcripts occur in many short-lived copies, the few cellular DNA molecules are long-lived. Hence pressures for functional swinger DNAs are greater than for swinger RNAs. Protein coding properties of swinger sequences differ from original sequences, suggesting rarity of corresponding swinger DNA. For genes producing structural RNAs, such as tRNAs and rRNAs, three exchanges (AT, CG and AT+CG) conserve self-hybridization properties. All nuclear eukaryote swinger DNA sequences detected in GenBank are for rRNA genes assuming AT+CG exchanges. In brachyuran crabs, 25 species had AT+CG swinger 18S rDNA, all matching the reverse-exchanged version of regular 18S rDNA of a related species. In this taxon, swinger replication of 18S rDNA apparently associated with, or even resulted in species radiation. AT+CG transformation doesn't invert sequence direction, differing from inverted repeats. Swinger repeats (detectable only assuming swinger transformations, AT+CG swinger repeats most frequent) within regular human rRNAs, independently confirm swinger polymerizations for most swinger types. Swinger replication might be an unsuspected molecular mechanism for ultrafast speciation.

  16. 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.

  17. 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


    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.

  18. 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.

  19. Single nucleotide polymorphism-based validation of exonic splicing enhancers.

    Directory of Open Access Journals (Sweden)

    William G Fairbrother


    Full Text Available Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality, which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE-ESE method. Aligning the single nucleotide polymorphisms (SNPs from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 +/- 0.05. This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype.

  20. Factor VII activating protease. Single nucleotide polymorphisms light the way. (United States)

    Kanse, S M; Etscheid, M


    Factor VII activating protease (FSAP) is a circulating serine protease with high homology to fibrinolytic enzymes. A role in the regulation of coagulation and fibrinolysis is suspected based on in vitro studies demonstrating activation of FVII or pro-urokinase plasminogen activator (uPA). However, considering the paucity of any studies in animal models or any correlative studies in humans the role of FSAP in haemostasis remains unclear. In relation to vascular remodeling processes or inflammation it has been convincingly shown that FSAP interacts with growth factors as well as protease activated receptors (PAR). Against this sparse background there are a plethora of studies which have investigated the linkage of single nucleotide polymorphisms (SNP) in the FSAP gene (HABP2) to various diseases. The G534E SNP of FSAP is associated with a low proteolytic activity due to an amino acid exchange in the protease domain. This and other SNPs have been linked to carotid stenosis, stroke as well as thrombosis in the elderly and plaque calcification. These SNP analyses indicate an important role for FSAP in the regulation of the haemostasis system as well as fibroproliferative inflammatory processes.

  1. 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


    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

  2. Phosphodiesterases: Regulators of cyclic nucleotide signals and novel molecular target for movement disorders. (United States)

    Sharma, Sorabh; Kumar, Kushal; Deshmukh, Rahul; Sharma, Pyare Lal


    Movement disorders rank among the most common neurological disorders. During the last two decades substantial progress has been made in understanding of the pathological basis of these disorders. Although, several mechanisms have been proposed, downregulation of cyclic nucleotide mediated signaling cascade has consistently been shown to contribute to the striatal dysfunctioning as seen in movement disorders. Thus, counteracting dysregulated cyclic nucleotide signaling has been considered to be beneficial in movement disorders. Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes responsible for the breakdown of cyclic nucleotides and upregulation in PDE activity has been reported in various movement disorders. Thus, PDE inhibition is considered to be a novel strategy to restore cerebral cyclic nucleotide levels and their downstream signalling cascade. Indeed, various PDE inhibitors have been tested pre-clinically and were reported to be neuroprotective in various neurodegenerative disorders associated with movement disabilities. In this review, we have discussed a putative role of PDE inhibitors in movement disorders and associated abnormalities.

  3. Complete nucleotide sequence of a monopartite Begomovirus and associated satellites infecting Carica papaya in Nepal. (United States)

    Shahid, M S; Yoshida, S; Khatri-Chhetri, G B; Briddon, R W; Natsuaki, K T


    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.

  4. Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.

    Directory of Open Access Journals (Sweden)

    Daniel Reinecke

    Full Text Available As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP and guanosine 3',5'-cyclic monophosphate (cGMP play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP and uridine 3',5'-cyclic monophosphate (cUMP also act as second messengers. Hydrolysis by phosphodiesterases (PDEs is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.

  5. Structure of the nucleotide complex of PyrR, the pyr attenuation protein from Bacillus caldolyticus, suggests dual regulation by pyrimidine and purine nucleotides. (United States)

    Chander, Preethi; Halbig, Kari M; Miller, Jamie K; Fields, Christopher J; Bonner, Heather K S; Grabner, Gail K; Switzer, Robert L; Smith, Janet L


    PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60 degrees C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg2+, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg2+ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.

  6. Single nucleotide polymorphism mining and nucleotide sequence analysis of Mx1 gene in exonic regions of Japanese quail

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    Diwesh Kumar Niraj


    Full Text Available Aim: An attempt has been made to study the Myxovirus resistant (Mx1 gene polymorphism in Japanese quail. Materials and Methods: In the present, investigation four fragments viz. Fragment I of 185 bp (Exon 3 region, Fragment II of 148 bp (Exon 5 region, Fragment III of 161 bp (Exon 7 region, and Fragment IV of 176 bp (Exon 13 region of Mx1 gene were amplified and screened for polymorphism by polymerase chain reaction-single-strand conformation polymorphism technique in 170 Japanese quail birds. Results: Out of the four fragments, one fragment (Fragment II was found to be polymorphic. Remaining three fragments (Fragment I, III, and IV were found to be monomorphic which was confirmed by custom sequencing. Overall nucleotide sequence analysis of Mx1 gene of Japanese quail showed 100% homology with common quail and more than 80% homology with reported sequence of chicken breeds. Conclusion: The Mx1 gene is mostly conserved in Japanese quail. There is an urgent need of comprehensive analysis of other regions of Mx1 gene along with its possible association with the traits of economic importance in Japanese quail.

  7. ACG: rapid inference of population history from recombining nucleotide sequences

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    O'Fallon Brendan D


    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.

  8. Sequencing genes in silico using single nucleotide polymorphisms

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    Zhang Xinyi


    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

  9. Analysis of codon usage and nucleotide composition bias in polioviruses

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    Gu Yuan-xing


    Full Text Available Abstract Background Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and a member of the family of Picornaviridae and among the most rapidly evolving viruses known. Analysis of codon usage can reveal much about the molecular evolution of the viruses. However, little information about synonymous codon usage pattern of polioviruses genome has been acquired to date. Methods The relative synonymous codon usage (RSCU values, effective number of codon (ENC values, nucleotide contents and dinucleotides were investigated and a comparative analysis of codon usage pattern for open reading frames (ORFs among 48 polioviruses isolates including 31 of genotype 1, 13 of genotype 2 and 4 of genotype 3. Results The result shows that the overall extent of codon usage bias in poliovirus samples is low (mean ENC = 53.754 > 40. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in those polioviruses. Depending on the RSCU data, it was found that there was a significant variation in bias of codon usage among three genotypes. Geographic factor also has some effect on the codon usage pattern (exists in the genotype-1 of polioviruses. No significant effect in gene length or vaccine derived polioviruses (DVPVs, wild viruses and live attenuated virus was observed on the variations of synonymous codon usage in the virus genes. The relative abundance of dinucleotide (CpG in the ORFs of polioviruses are far below expected values especially in DVPVs and attenuated virus of polioviruses genotype 1. Conclusion The information from this study may not only have theoretical value in understanding poliovirus evolution, especially for DVPVs genotype 1, but also have potential value for the development of poliovirus vaccines.

  10. 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


    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.

  11. tRNA nucleotide 47: an evolutionary enigma. (United States)

    Cermakian, N; McClain, W H; Cedergren, R


    A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.

  12. Network analysis of single nucleotide polymorphisms in asthma (United States)

    Renkonen, Jutta; Joenväärä, Sakari; Parviainen, Ville; Mattila, Pirkko; Renkonen, Risto


    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

  13. Association of prediabetes-associated single nucleotide polymorphisms with microalbuminuria (United States)

    Choi, Jong Wook; Moon, Shinje; Jang, Eun Jung; Lee, Chang Hwa; Park, Joon-Sung


    Increased glycemic exposure, even below the diagnostic criteria for diabetes mellitus, is crucial in the pathogenesis of diabetic microvascular complications represented by microalbuminuria. Nonetheless, there is limited evidence regarding which single nucleotide polymorphisms (SNPs) are associated with prediabetes and whether genetic predisposition to prediabetes is related to microalbuminuria, especially in the general population. Our objective was to answer these questions. We conducted a genomewide association study (GWAS) separately on two population-based cohorts, Ansung and Ansan, in the Korean Genome and Epidemiology Study (KoGES). The initial GWAS was carried out on the Ansung cohort, followed by a replication study on the Ansan cohort. A total of 5682 native Korean participants without a significant medical illness were classified into either control group (n = 3153) or prediabetic group (n = 2529). In the GWAS, we identified two susceptibility loci associated with prediabetes, one at 17p15.3-p15.1 in the GCK gene and another at 7p15.1 in YKT6. When variations in GCK and YKT6 were used as a model of prediabetes, this genetically determined prediabetes increased microalbuminuria. Multiple logistic regression analyses revealed that fasting glucose concentration in plasma and SNP rs2908289 in GCK were associated with microalbuminuria, and adjustment for age, gender, smoking history, systolic blood pressure, waist circumference, and serum triglyceride levels did not attenuate this association. Our results suggest that prediabetes and the associated SNPs may predispose to microalbuminuria before the diagnosis of diabetes mellitus. Further studies are needed to explore the details of the physiological and molecular mechanisms underlying this genetic association. PMID:28158221

  14. Old concepts, new molecules and current approaches applied to the bacterial nucleotide signalling field (United States)


    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

  15. 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


    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.

  16. 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


    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

  17. Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane (United States)

    Townsend, Michelle H; Anderson, Michael D; Weagel, Evita G; Velazquez, Edwin J; Weber, K Scott; Robison, Richard A; O’Neill, Kim L


    In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the

  18. Does the G.G*syn DNA mismatch containing canonical and rare tautomers of the guanine tautomerise through the DPT? A QM/QTAIM microstructural study (United States)

    Brovarets', Ol'ha O.; Hovorun, Dmytro M.


    We have established that the asynchronous concerted double proton transfer (DPT), moving with a time gap and without stable intermediates, is the underlying mechanism for the tautomerisation of the G.G*syn DNA base mispair (C1 symmetry), formed by the keto and enol tautomers of the guanine in the anti- and syn-configurations, into the G*.G*syn base mispair (C1), formed by the enol and imino tautomers of the G base, using quantum-mechanical calculations and Bader's quantum theory of atoms in molecules. By constructing the sweeps of the geometric, electron-topological, energetic, polar and natural bond orbital properties along the intrinsic reaction coordinate of the G.G*syn↔G*.G*syn DPT tautomerisation, the nine key points, that are critical for the atomistic understanding of the tautomerisation reaction, were set and comprehensively analysed. It was found that the G.G*syn mismatch possesses pairing scheme with the formation of the O6...HO6 (7.01) and N1H...N7 (6.77) H-bonds, whereas the G*.G*syn mismatch - of the O6H...O6 (10.68) and N1...HN7 (9.59 kcal mol-1) H-bonds. Our results highlight that these H-bonds are significantly cooperative and mutually reinforce each other in both mismatches. The deformation energy necessary to apply for the G.G*syn base mispair to acquire the Watson-Crick sizes has been calculated. We have shown that the thermodynamically stable G*.G*syn base mispair is dynamically unstable structure with a lifetime of 4.1 × 10-15 s and any of its six low-lying intermolecular vibrations can develop during this period of time. These data exclude the possibility to change the tautomeric status of the bases under the dissociation of the G.G*syn mispair into the monomers during DNA replication. Finally, it has been made an attempt to draw from the physico-chemical properties of all four incorrect purine-purine DNA base pairs a general conclusion, which claims the role of the transversions in spontaneous point mutagenesis.

  19. Dietary Nucleotides Supplementation and Liver Injury in Alcohol-Treated Rats: A Metabolomics Investigation

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    Xiaxia Cai


    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.

  20. Changes of cytokinin nucleotides in an anise cell culture (Pimpinella anisum L.) during growth and embryogenesis. (United States)

    Ernst, D; Oesterhelt, D


    Endogenous levels of cytokinin nucleotides in an anise cell culture were determined during proembryonal, as well as embryonal development. In both cultures the maximum level of isopentenyladenine nucleotides was found during the first four days of incubation which correlated with the beginning of logarithmic growth (embryonal: 8 ng g(-1) tresh weight; proembryonal: 17.4 ng g(-1) fresh weight). The concentration of zeatin nucleotides remained constant at a very low level. The present data and those of Ernst et al. (1984) and Ernst and Oesterhelt (1984) are concerned in ascribing a major role to cytokinins in cell division, but not in embryo differentiation.

  1. Effects of six anaesthetic agents on UDP-glucuronic acid and other nucleotides in rat liver. (United States)

    Christensson, P I; Eriksson, G


    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.

  2. Predicting protein-binding RNA nucleotides using the feature-based removal of data redundancy and the interaction propensity of nucleotide triplets. (United States)

    Choi, Sungwook; Han, Kyungsook


    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

  3. Myosin individualized: single nucleotide polymorphisms in energy transduction

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    Wieben Eric D


    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

  4. Single nucleotide polymorphism discovery in elite north american potato germplasm

    Directory of Open Access Journals (Sweden)

    De Jong Walter S


    Full Text Available Abstract Background Current breeding approaches in potato rely almost entirely on phenotypic evaluations; molecular markers, with the exception of a few linked to disease resistance traits, are not widely used. Large-scale sequence datasets generated primarily through Sanger Expressed Sequence Tag projects are available from a limited number of potato cultivars and access to next generation sequencing technologies permits rapid generation of sequence data for additional cultivars. When coupled with the advent of high throughput genotyping methods, an opportunity now exists for potato breeders to incorporate considerably more genotypic data into their decision-making. Results To identify a large number of Single Nucleotide Polymorphisms (SNPs in elite potato germplasm, we sequenced normalized cDNA prepared from three commercial potato cultivars: 'Atlantic', 'Premier Russet' and 'Snowden'. For each cultivar, we generated 2 Gb of sequence which was assembled into a representative transcriptome of ~28-29 Mb for each cultivar. Using the Maq SNP filter that filters read depth, density, and quality, 575,340 SNPs were identified within these three cultivars. In parallel, 2,358 SNPs were identified within existing Sanger sequences for three additional cultivars, 'Bintje', 'Kennebec', and 'Shepody'. Using a stringent set of filters in conjunction with the potato reference genome, we identified 69,011 high confidence SNPs from these six cultivars for use in genotyping with the Infinium platform. Ninety-six of these SNPs were used with a BeadXpress assay to assess allelic diversity in a germplasm panel of 248 lines; 82 of the SNPs proved sufficiently informative for subsequent analyses. Within diverse North American germplasm, the chip processing market class was most distinct, clearly separated from all other market classes. The round white and russet market classes both include fresh market and processing cultivars. Nevertheless, the russet and round

  5. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data. (United States)


    ... 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...

  6. Synthesis of PET probe O(6)-[(3-[(11)C]methyl)benzyl]guanine by Pd(0)-mediated rapid C-[(11)C]methylation toward imaging DNA repair protein O(6)-methylguanine-DNA methyltransferase in glioblastoma. (United States)

    Koyama, Hiroko; Ikenuma, Hiroshi; Toda, Hiroshi; Kondo, Goro; Hirano, Masaki; Kato, Masaya; Abe, Junichiro; Yamada, Takashi; Wakabayashi, Toshihiko; Ito, Kengo; Natsume, Atsushi; Suzuki, Masaaki


    O(6)-Benzylguanine (O(6)-BG) is a substrate of O(6)-methylguanine-DNA methyltransferase (MGMT), which is involved in drug resistance of chemotherapy in the majority of glioblastoma multiform. For clinical diagnosis, it is hoped that the MGMT expression level could be determined by a noninvasive method to understand the detailed biological properties of MGMT-specific tumors. We synthesized (11)C-labeled O(6)-[(3-methyl)benzyl]guanine ([(11)C]mMeBG) as a positron emission tomography probe. Thus, a mixed amine-protected stannyl precursor, N(9)-(tert-butoxycarbonyl)-O(6)-[3-(tributylstannyl)benzyl]-N(2)-(trifluoroacetyl)guanine, was subjected to rapid C-[(11)C]methylation under [(11)C]CH3I/[Pd2(dba)3]/P(o-CH3C6H4)3/CuCl/K2CO3 in NMP, followed by quick deprotection with LiOH/H2O, giving [(11)C]mMeBG with total radioactivity of 1.34GBq and ≥99% radiochemical and chemical purities.

  7. The Label-Free Unambiguous Detection and Symbolic Display of Single Nucleotide Polymorphisms on DNA Origami (United States)

    Subramanian, Hari K. K.; Chakraborty, Banani; Sha, Ruojie; Seeman, Nadrian C.


    Single Nucleotide Polymorphisms (SNPs) are the most common genetic variation in the human genome. Kinetic methods based on branch migration have proved successful for detecting SNPs because a mispair inhibits the progress of branch migration in the direction of the mispair. We have combined the effectiveness of kinetic methods with AFM of DNA origami patterns to produce a direct visual readout of the target nucleotide contained in the probe sequence. The origami contains graphical representations of the four nucleotide alphabetic characters, A, T, G and C, and the symbol containing the test nucleotide identity vanishes in the presence of the probe. The system also works with pairs of probes, corresponding to heterozygous diploid genomes. PMID:21235216

  8. Mayaro virus: complete nucleotide sequence and phylogenetic relationships with other alphaviruses. (United States)

    Lavergne, Anne; de Thoisy, Benoît; Lacoste, Vincent; Pascalis, Hervé; Pouliquen, Jean-François; Mercier, Véronique; Tolou, Hugues; Dussart, Philippe; Morvan, Jacques; Talarmin, Antoine; Kazanji, Mirdad


    Mayaro (MAY) virus is a member of the genus Alphavirus in the family Togaviridae. Alphaviruses are distributed throughout the world and cause a wide range of diseases in humans and animals. Here, we determined the complete nucleotide sequence of MAY from a viral strain isolated from a French Guianese patient. The deduced MAY genome was 11,429 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. Nucleotide and amino acid homologies, as well as phylogenetic analyses of the obtained sequence confirmed that MAY is not a recombinant virus and belongs to the Semliki Forest complex according to the antigenic complex classification. Furthermore, analyses based on the E1 region revealed that MAY is closely related to Una virus, the only other South American virus clustering with the Old World viruses. On the basis of our results and of the alphaviruses diversity and pathogenicity, we suggest that alphaviruses may have an Old World origin.

  9. Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids. (United States)

    Gissot, Arnaud; Camplo, Michel; Grinstaff, Mark W; Barthélémy, Philippe


    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.

  10. Nucleotide sequences of two Korean isolates of Cucumber green mottle mosaic virus. (United States)

    Kim, Sang-Min; Lee, Jung-Myung; Yim, Kyu-Ock; Oh, Man-Ho; Park, Jin-Woo; Kim, Kook-Hyung


    The nucleotide sequences of the genomic RNAs of Cucumber green mottle mosaic virus Korean watermelon isolate (CGMMV-KW) and Korean oriental melon isolate (CGMMV-KOM) were determined and compared to the sequences of other tobamoviruses including CGMMV strains W and SH. Each CGMMV isolate had a genome of 6,424 nucleotides. Each also had 60 and 176 nucleotides of 5' and 3' untranslated regions (UTRs), respectively, and four open reading frames (ORF1-4). ORFs 1 to 4 encode proteins of 129, 186, 29, and 17.4 kDa, respectively. The nucleotide and deduced amino acid sequences of CGMMV-KOM and CGMMV-KW were more than 98.3% identical. When compared to other CGMMV strains in a phylogenetic analysis they were found to form a distinct virus clade, and were more distantly related to other tobamoviruses (23.5-56.7% identity).

  11. 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.


    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.

  12. Organization of Nucleotides in Different Environments and the Formation of Pre-Polymers


    Sebastian Himbert; Mindy Chapman; Deamer, David W.; Rheinstädter, Maikel C.


    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, nan...

  13. 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


    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.

  14. The Immunosuppressant Mycophenolic Acid Alters Nucleotide and Lipid Metabolism in an Intestinal Cell Model (United States)

    Heischmann, Svenja; Dzieciatkowska, Monika; Hansen, Kirk; Leibfritz, Dieter; Christians, Uwe


    The study objective was to elucidate the molecular mechanisms underlying the negative effects of mycophenolic acid (MPA) on human intestinal cells. Effects of MPA exposure and guanosine supplementation on nucleotide concentrations in LS180 cells were assessed using liquid chromatography-mass spectrometry. Proteomics analysis was carried out using stable isotope labeling by amino acids in cell culture combined with gel-based liquid chromatography-mass spectrometry and lipidome analysis using 1H nuclear magnetic resonance spectroscopy. Despite supplementation, depletion of guanosine nucleotides (p < 0.001 at 24 and 72 h; 5, 100, and 250 μM MPA) and upregulation of uridine and cytidine nucleotides (p < 0.001 at 24 h; 5 μM MPA) occurred after exposure to MPA. MPA significantly altered 35 proteins mainly related to nucleotide-dependent processes and lipid metabolism. Cross-reference with previous studies of MPA-associated protein changes widely corroborated these results, but showed differences that may be model- and/or method-dependent. MPA exposure increased intracellular concentrations of fatty acids, cholesterol, and phosphatidylcholine (p < 0.01 at 72 h; 100 μM MPA) which corresponded to the changes in lipid-metabolizing proteins. MPA affected intracellular nucleotide levels, nucleotide-dependent processes, expression of structural proteins, fatty acid and lipid metabolism in LS180 cells. These changes may compromise intestinal membrane integrity and contribute to gastrointestinal toxicity. PMID:28327659

  15. Organization of Nucleotides in Different Environments and the Formation of Pre-Polymers (United States)

    Himbert, Sebastian; Chapman, Mindy; Deamer, David W.; Rheinstädter, Maikel C.


    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.

  16. NextSearch: A Search Engine for Mass Spectrometry Data against a Compact Nucleotide Exon Graph. (United States)

    Kim, Hyunwoo; Park, Heejin; Paek, Eunok


    Proteogenomics research has been using six-frame translation of the whole genome or amino acid exon graphs to overcome the limitations of reference protein sequence database; however, six-frame translation is not suitable for annotating genes that span over multiple exons, and amino acid exon graphs are not convenient to represent novel splice variants and exon skipping events between exons of incompatible reading frames. We propose a proteogenomic pipeline NextSearch (Nucleotide EXon-graph Transcriptome Search) that is based on a nucleotide exon graph. This pipeline consists of constructing a compact nucleotide exon graph that systematically incorporates novel splice variations and a search tool that identifies peptides by directly searching the nucleotide exon graph against tandem mass spectra. Because our exon graph stores nucleotide sequences, it can easily represent novel splice variations and exon skipping events between incompatible reading frame exons. Searching for peptide identification is performed against this nucleotide exon graph, without converting it into a protein sequence in FASTA format, achieving an order of magnitude reduction in the size of the sequence database storage. NextSearch outputs the proteome-genome/transcriptome mapping results in a general feature format (GFF) file, which can be visualized by public tools such as the UCSC Genome Browser.

  17. Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs. (United States)

    Hacker, Stephan M; Buntz, Annette; Zumbusch, Andreas; Marx, Andreas


    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.

  18. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides. (United States)

    Lehninger, A L; Vercesi, A; Bababunmi, E A


    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.

  19. 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


    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.

  20. 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. (United States)

    Silva-Junior, Orzenil B; Grattapaglia, Dario


    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.

  1. Association of Toll-like receptor 2 Arg753Gln and Toll-like receptor 1 Ile602Ser single-nucleotide polymorphisms with leptospirosis in an Argentine population. (United States)

    Cédola, Maia; Chiani, Yosena; Pretre, Gabriela; Alberdi, Lucrecia; Vanasco, Bibiana; Gómez, Ricardo M


    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.

  2. Multifactor dimensionality reduction analysis identifies specific nucleotide patterns promoting genetic polymorphisms

    Directory of Open Access Journals (Sweden)

    Arehart Eric


    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 (United States)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Watkins, C. L.; Hall, L. M.


    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. Comprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing. (United States)

    Rutherford, Steven T; Valastyan, Julie S; Taillefumier, Thibaud; Wingreen, Ned S; Bassler, Bonnie L


    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.

  5. Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry. (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


    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. On the role of a coumarin derivative for sensing applications: Nucleotide identification using a micellar system. (United States)

    Bettoschi, Alexandre; Ceglie, Andrea; Lopez, Francesco; Meli, Valeria; Murgia, Sergio; Tamburro, Manuela; Caltagirone, Claudia; Cuomo, Francesca


    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.

  7. Sublingual Nucleotides Prolong Run Time to Exhaustion in Young Physically Active Men

    Directory of Open Access Journals (Sweden)

    Sergej M. Ostojic


    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.

  8. Nucleotide precursors prevent folic acid-resistant neural tube defects in the mouse. (United States)

    Leung, Kit-Yi; De Castro, Sandra C P; Savery, Dawn; Copp, Andrew J; Greene, Nicholas D E


    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.

  9. Nucleotide composition of CO1 sequences in Chelicerata (Arthropoda): detecting new mitogenomic rearrangements. (United States)

    Arabi, Juliette; Judson, Mark L I; Deharveng, Louis; Lourenço, Wilson R; Cruaud, Corinne; Hassanin, Alexandre


    Here we study the evolution of nucleotide composition in third codon-positions of CO1 sequences of Chelicerata, using a phylogenetic framework, based on 180 taxa and three markers (CO1, 18S, and 28S rRNA; 5,218 nt). The analyses of nucleotide composition were also extended to all CO1 sequences of Chelicerata found in GenBank (1,701 taxa). The results show that most species of Chelicerata have a positive strand bias in CO1, i.e., in favor of C nucleotides, including all Amblypygi, Palpigradi, Ricinulei, Solifugae, Uropygi, and Xiphosura. However, several taxa show a negative strand bias, i.e., in favor of G nucleotides: all Scorpiones, Opisthothelae spiders and several taxa within Acari, Opiliones, Pseudoscorpiones, and Pycnogonida. Several reversals of strand-specific bias can be attributed to either a rearrangement of the control region or an inversion of a fragment containing the CO1 gene. Key taxa for which sequencing of complete mitochondrial genomes will be necessary to determine the origin and nature of mtDNA rearrangements involved in the reversals are identified. Acari, Opiliones, Pseudoscorpiones, and Pycnogonida were found to show a strong variability in nucleotide composition. In addition, both mitochondrial and nuclear genomes have been affected by higher substitution rates in Acari and Pseudoscorpiones. The results therefore indicate that these two orders are more liable to fix mutations of all types, including base substitutions, indels, and genomic rearrangements.

  10. Identification of Single Nucleotide Polymorphism on Growth Hormone Gene in Aceh Cattle

    Directory of Open Access Journals (Sweden)

    E. M. Sari


    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.

  11. Prebiotic synthesis of nucleotides at the Earth orbit in presence of Lunar soil. (United States)

    Kuzicheva, E A; Gontareva, N B


    Modern studies now favor the fact that extraterrestrial organic molecules served as an important source of biological important substances on the primitive Earth. It is presumed that these space-made organic molecules could be transported safely to the Earth surface being associated with mineral grains. It is important to test whether nucleotides synthesized in Earth orbit could be protected by lunar surface regolite. The phosphorylation of adenosine, uridine and thymidine has been studied with respect of their further transformations and degradation in presence of mineral bed. After retrieval, HPLC analysis is used to identify all the mononucleotides of certain nucleosides. It has been shown, that exposure of the investigated nucleosides as dry films in space conditions in the presence of Lunar soil increases the yield of synthesized nucleotides in 1.1-3.0 times as compared with the exposure of the same samples in absence of Lunar soil. To identify and evaluate the principal source of energy in open space responsible for nucleotide synthesis reaction laboratory experiments were performed. It has been shown, that vacuum ultra violet (VUV 145 nm) radiation promotes nucleotide synthesis more effectively than ultra violet (UV 254 nm) while the presence of Lunar soil increases reaction yield in 1.5-2.0 times. Formation of 5'-mononucleotides seemed to be the most effective reaction both in flight and in laboratory experiments. Protective action of lunar soil on synthesized nucleotides against UV radiation has been shown in open Space conditions.

  12. Remarkable similarity in genome nucleotide sequences between the Schwarz FF-8 and AIK-C measles virus vaccine strains and apparent nucleotide differences in the phosphoprotein gene. (United States)

    Ito, Chie; Ohgimoto, Shinji; Kato, Seiichi; Sharma, Luna Bhatta; Ayata, Minoru; Komase, Katsuhiro; Takeuchi, Kaoru; Ihara, Toshiaki; Ogura, Hisashi


    The Schwarz FF-8 (FF-8) and AIK-C measles virus vaccine strains are currently used for vaccination in Japan. Here, the complete genome nucleotide sequence of the FF-8 strain has been determined and its genome sequence found to be remarkably similar to that of the AIK-C strain. These two strains are differentiated only by two nucleotide differences in the phosphoprotein gene. Since the FF-8 strain does not possess the amino acid substitutions in the phospho- and fusion proteins which are responsible for the temperature-sensitivity and small syncytium formation phenotypes of the AIK-C strain, respectively, other unidentified common mechanisms likely attenuate both the FF-8 and AIK-C strains.

  13. Structural basis for dual nucleotide selectivity of aminoglycoside 2''-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases. (United States)

    Shi, Kun; Berghuis, Albert M


    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.

  14. Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells. (United States)

    Guo, Jia; Hanawalt, Philip C; Spivak, Graciela


    Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

  15. IRE1α nucleotide sequence cleavage specificity in the unfolded protein response. (United States)

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J; Tirasophon, Witoon


    Inositol-requiring enzyme 1 (IRE1) is a conserved sensor of the unfolded protein response that has protein kinase and endoribonuclease (RNase) enzymatic activities and thereby initiates HAC1/XBP1 splicing. Previous studies demonstrated that human IRE1α (hIRE1α) does not cleave Saccharomyces cerevisiae HAC1 mRNA. Using an in vitro cleavage assay, we show that adenine to cytosine nucleotide substitution at the +1 position in the 3' splice site of HAC1 RNA is required for specific cleavage by hIRE1α. A similar restricted nucleotide specificity in the RNA substrate was observed for XBP1 splicing in vivo. Together these findings underscore the essential role of cytosine nucleotide at +1 in the 3' splice site for determining cleavage specificity of hIRE1α.

  16. Complete nucleotide sequence of the new potexvirus "Alstroemeria virus X". Brief report. (United States)

    Fuji, S; Shinoda, K; Ikeda, M; Furuya, H; Naito, H; Fukumoto, F


    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).

  17. Complete Nucleotide Sequence of a Newly Avirulent Newcastle Disease Virus Hubei 92(HB92) Strain

    Institute of Scientific and Technical Information of China (English)

    Pan Zi-shu; Chen Yu-dong; Shao Hua-bin; Yang Jun; Xiong Zhong-liang; Wen Guo-yuan; Zhang Chu-yu


    A new avirulent, heat-resistance HB92 strain of newcastle Disease Virus (NDV) was acquired from Australia V4 strain. Its complete nucleotides sequence was first determined. The entire genome of NDV HB92 consists of 15 186nucleotides (GenBank accession no. AY225110 ). It was demonstrated by sequence analysis that nucleotides homology of HB92 strain with virulent strain ZJ1 were 83.9%, and the homology compared with avirulent vaccine strain La Sota and BI were 94. 0% and 93. 5%, respectively. These results might be contributive to tbe study of the molecular mechanism of evolution of the NDV strain HB92 and to develop the engineered recombinant vaccine.

  18. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay. (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M


    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.

  19. Study of the Molecular Recognition of Nucleotides and Bases by a Novel Calixarene Derivative Containing Uracil

    Institute of Scientific and Technical Information of China (English)

    SHI,Hui-Jie; SHI,Xian-Fa; YAO,Tian-Ming; JI,Liang-Nian


    A calix[4]arene derivative containing uracil, 5-(uracil-N1-acetamido)-25,26,27,28-tetrahy droxycalix[4]-arene (UC), was designed and synthesized. The interaction with nucleotides and bases has also been studied by ESI-MS and π-A isotherms. The results of ESI-MS showed that UC could recognize adenine and adenosine from other nucleotides and bases. In addition, π-A isotherms at the air-water interface indicated that there was interaction between UC and the species in the subphase, and the respective complexes were formed in the monolayer. The mean molecular area at zero surface pressure increased with the sizes of the nucleotides and bases in the subphase in the order: water<adenine<adenosine<ATP·Na2.

  20. Characterization of single nucleotide polymorphism markers for the green sea turtle (Chelonia mydas). (United States)

    Roden, Suzanne E; Dutton, Peter H; Morin, Phillip A


    We present data on 29 new single nucleotide polymorphism assays for the green sea turtle, Chelonia mydas. DNA extracts from 39 green turtles were used for two methods of single nucleotide polymorphism discovery. The first approach employed an amplified fragment length polymorphism technique. The second technique screened a microsatellite library. Allele-specific amplification assays were developed for high-throughput single nucleotide polymorphism genotyping and tested on two Pacific C. mydas nesting populations. Observed heterozygosities ranged from 0 to 0.95 for a Hawaiian population and from 0 to 0.85 for a Galapagos population. Each of the populations had one locus out of Hardy-Weinberg equilibrium, SSCM2b and SSCM5 for Hawaii and Galapagos, respectively. No loci showed significant genotypic linkage disequilibrium across an expanded set of four Pacific nesting populations. However, two loci, SSCM4 and SSCM10b showed linkage disequilibrium across three populations indicating possible association.

  1. Crystal structure of cyclic nucleotide-binding-like protein from Brucella abortus. (United States)

    He, Zheng; Gao, Yuan; Dong, Jing; Ke, Yuehua; Li, Xuemei; Chen, Zeliang; Zhang, Xuejun C


    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.

  2. Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina. (United States)

    Trucco, Verónica; de Breuil, Soledad; Bejerman, Nicolás; Lenardon, Sergio; Giolitti, Fabián


    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.

  3. Associations between single nucleotide polymorphisms in multiple candidate genes and body weight in rabbits (United States)

    El-Sabrout, Karim; Aggag, Sarah A.


    Aim: In this study, we examined parts of six growth genes (growth hormone [GH], melanocortin 4 receptor [MC4R], growth hormone receptor [GHR], phosphorglycerate mutase [PGAM], myostatin [MSTN], and fibroblast growth factor [FGF]) as specific primers for two rabbit lines (V-line, Alexandria) using nucleotide sequence analysis, to investigate association between detecting single nucleotide polymorphism (SNP) of these genes and body weight (BW) at market. Materials and Methods: Each line kits were grouped into high and low weight rabbits to identify DNA markers useful for association studies with high BW. DNA from blood samples of each group was extracted to amplify the six growth genes. SNP technique was used to study the associate polymorphism in the six growth genes and marketing BW (at 63 days) in the two rabbit lines. The purified polymerase chain reaction products were sequenced in those had the highest and lowest BW in each line. Results: Alignment of sequence data from each group revealed the following SNPs: At nucleotide 23 (A-C) and nucleotide 35 (T-G) in MC4R gene (sense mutation) of Alexandria and V-line high BW. Furthermore, we detected the following SNPs variation between the two lines: A SNP (T-C) at nucleotide 27 was identified by MC4R gene (sense mutation) and another one (A-C) at nucleotide 14 was identified by GHR gene (nonsense mutation) of Alexandria line. The results of individual BW at market (63 days) indicated that Alexandria rabbits had significantly higher BW compared with V-line rabbits. MC4R polymorphism showed significant association with high BW in rabbits. Conclusion: The results of polymorphism demonstrate the possibility to detect an association between BW in rabbits and the efficiency of the used primers to predict through the genetic specificity using the SNP of MC4R. PMID:28246458

  4. Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9. (United States)

    Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis


    The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.

  5. Complete nucleotide sequences of two adjacent early vaccinia virus genes located within the inverted terminal repetition. (United States)

    Venkatesan, S; Gershowitz, A; Moss, B


    The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol. 65:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine . thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3' end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3' ends of the two transcripts were located within 100 bp of each other. Although there are adenine . thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.

  6. Studies of Hepatic Lesion on Hyperuricemia Rat-model Induced by Adenine and Guanine%腺嘌呤、鸟嘌呤致高尿酸血症大鼠肝脏损害的研究

    Institute of Scientific and Technical Information of China (English)

    宋燕郡; 于维森; 殷凡; 宋扬


    Objective:To observe adenine and guanine effect liver function of hyperuricemia-model rat, and the changes under ultra microstructure.Method:The 36 male Wistar rats were randomly divided into 6 groups (A-control group,B-model group,C-adenine group,D-adenine starch paste group,E-adenine and guanine group,F-guanine group),each group of 6 cases.Groups B, C, D, E, F continuously given Yeast Extract Solution 15 g/(kg·d) to fill the stomach,7 days induced hyperuricemia-model rats.After the success of the building, group B for starch paste to fill the stomach.Group C:20 mg/(kg·d) adenine starch paste mixture.Group D:10 mg/(kg·d) adenine starch paste mixture.Group E:10 mg/(kg·d) adenine mixed 10 mg/(kg·d) guanine starch suspension.Group F:20 mg/(kg·d) guanine starch paste mixture to fill the stomach.For 14 days,the determination serum ALT,AST,UA and liver tissue electron microscope observation of liver injury of each group rats.Result:(1)Ultra microstructure observation the lysosome of group C increased obviously,scattered around the nucleus,and had dark grain material.Group D lysosome amount increased slightly,increased lipid drops, enter the lysosome.Group E the amount of lysosome increasing, increased lipid droplets, appear dark granular material.Group F bile duct in a small dark granular material.(2)ALT,AST of C-F groups were compared with group B,the differences were statistically significant(P cause ALT and AST levels significantly in hyperuricemia-model rats, the effect is higher than the same dose of guanine.Adenine has obvious effect to rat blood uric acid levels.%目的:观察腺嘌呤、鸟嘌呤作用高尿酸血症大鼠肝脏时,肝脏功能变化情况及透射电镜下肝脏超微结构的变化。方法:选用实验用雄性Wistar大鼠36只,随机分为A组(对照组)、B组(造模对照组)、C组(腺嘌呤组)、D组(腺嘌呤淀粉糊组)、E组(腺嘌呤、鸟嘌呤组)、F组(鸟嘌呤组),每组6只。B、C

  7. Adenine nucleotide effect on intraocular pressure: Involvement of the parasympathetic nervous system. (United States)

    Peral, Assumpta; Gallar, Juana; Pintor, Jesús


    Nucleotides are present in the aqueous humor possibly exerting physiological effects on intraocular pressure (IOP). To determine the effect of nucleotides such as ATP and its related derivatives on IOP, New Zealand white rabbits were used. IOP was measured in rabbits treated topically either with saline (control) or with a single dose (10 microg/microL) of adenine nucleotides (ATP, 2-meS-ATP, ATP-gamma-S, alpha,beta-meADP, alpha,beta-meATP and beta,gamma-meATP). Those nucleotides reducing IOP (alpha,beta-meATP and beta,gamma-meATP) were then tested in concentrations ranging from 1 to 100 microg/microL to obtain the IC(50) value. Several antagonists for the P2 and adenosine A1 receptors (all at 10 microg/microL) were assayed 30 min before the application of the hypotensive nucleotide beta,gamma-meATP. To see whether the nucleotide was acting directly on the structures involved in aqueous humor dynamics or on the autonomic nerves controlling IOP, animal denervation and sympathetic (yohimbine and ICI-118,551 at 10 microg/microL) and parasympathetic (atropine and hexametonium at 10 microg/microL) receptors' antagonists were used 30 min before the instillation of beta,gamma-meATP. alpha,beta-meATP and beta,gamma-meATP decreased IOP to 60% of control value (basal IOP=23.2+/-1.3 mmHg), with IC(50) of 1.59+/-0.21 microg/microLand 0.56+/-0.62 microg/microL, which corresponds to 3mM and 1mM respectively. Denervation completely abolished the effect of beta,gamma-meATP. Sympathetic antagonists did not modify the hypotensive effect of beta,gamma-meATP, but parasympathetic antagonists were able to abolish it. Among the series of adenine nucleotide tested, alpha,beta-meATP and beta,gamma-meATP presented hypotensive actions on IOP. beta,gamma-meATP seems to stimulate cholinergic terminals being its final effect the IOP reduction. Therefore, these two nucleotides are interesting pharmacological tools for those pathologies related with high intraocular pressure.

  8. Glutamine and nucleotide supplementation in broiler diets in alternative breeding system


    Zavarize, K C [UNESP; Sartori,José Roberto; Pelícia, V C [UNESP; Pezzato, Antonio Celso [UNESP; Araujo, P C; Stradiotti, A C [UNESP; Madeira, L A [UNESP


    The experiment was conducted to evaluate the effect of supplementing glutamine and nucleotides on growth performance and development intestinal morphology in broiler chicks. In the trial, 600 male broiler chicks distributed in randomized blocks in a 3x2 factorial arrangement (consisting of a uniform basal diet supplemented with: 0.0, 0.5 or 1.0% glutamine, and 0.0 or 0.04% nucleotides), for a total of 6 treatments with 25 birds each. Means of performance (weight, weight gain, feed intake, fee...

  9. Dependence of nucleotide physical properties on their placement in codons and determinative degree

    Institute of Scientific and Technical Information of China (English)


    Various physical properties such as dipole moment, heat of formation and energy of the most stable formation of nucleotides and bases were calculated by PM3 (modified neglect of diatomic overlap, parametric method number 3) and AM1 (austin model 1) methods. As distinct from previous calculations, for nucleotides the interaction with neighbours is taken into account up to gradient of convergence equaling 1. The dependencies of these variables from the place in the codon and the determinative degree were obtained. The difference of these variables for codons and anticodons is shown.

  10. Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins. (United States)

    Bardaweel, Sanaa K; Ghosh, Brahma; Wagner, Carston R


    Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding Protein 1 (hHint1). Using our established phosphoramidase assay, K(i) values were determined. All compounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intracellular inhibitors of ecHinT.

  11. Rates of gene rearrangement and nucleotide substitution are correlated in the mitochondrial genomes of insects. (United States)

    Shao, Renfu; Dowton, Mark; Murrell, Anna; Barker, Stephen C


    A number of studies indicated that lineages of animals with high rates of mitochondrial (mt) gene rearrangement might have high rates of mt nucleotide substitution. We chose the hemipteroid assemblage and the Insecta to test the idea that rates of mt gene rearrangement and mt nucleotide substitution are correlated. For this purpose, we sequenced the mt genome of a lepidopsocid from the Psocoptera, the only order of hemipteroid insects for which an entire mtDNA sequence is not available. The mt genome of this lepidopsocid is circular, 16,924 bp long, and contains 37 genes and a putative control region; seven tRNA genes and a protein-coding gene in this genome have changed positions relative to the ancestral arrangement of mt genes of insects. We then compared the relative rates of nucleotide substitution among species from each of the four orders of hemipteroid insects and among the 20 insects whose mt genomes have been sequenced entirely. All comparisons among the hemipteroid insects showed that species with higher rates of gene rearrangement also had significantly higher rates of nucleotide substitution statistically than did species with lower rates of gene rearrangement. In comparisons among the 20 insects, where the mt genomes of the two species differed by more than five breakpoints, the more rearranged species always had a significantly higher rate of nucleotide substitution than the less rearranged species. However, in comparisons where the mt genomes of two species differed by five or less breakpoints, the more rearranged species did not always have a significantly higher rate of nucleotide substitution than the less rearranged species. We tested the statistical significance of the correlation between the rates of mt gene rearrangement and mt nucleotide substitution with nine pairs of insects that were phylogenetically independent from one another. We found that the correlation was positive and statistically significant (R2 = 0.73, P = 0.01; Rs = 0.67, P

  12. Effect of the nucleotides surrounding the start codon on the translation of foot-and-mouth disease virus RNA. (United States)

    Ma, X X; Feng, Y P; Gu, Y X; Zhou, J H; Ma, Z R


    As for the alternative AUGs in foot-and-mouth disease virus (FMDV), nucleotide bias of the context flanking the AUG(2nd) could be used as a strong signal to initiate translation. To determine the role of the specific nucleotide context, dicistronic reporter constructs were engineered to contain different versions of nucleotide context linking between internal ribosome entry site (IRES) and downstream gene. The results indicate that under FMDV IRES-dependent mechanism, the nucleotide contexts flanking start codon can influence the translation initiation efficiencies. The most optimal sequences for both start codons have proved to be UUU AUG(1st) AAC and AAG AUG(2nd) GAA.

  13. Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone. (United States)

    Amick, Joseph; Schlanger, Simon E; Wachnowsky, Christine; Moseng, Mitchell A; Emerson, Corey C; Dare, Michelle; Luo, Wen-I; Ithychanda, Sujay S; Nix, Jay C; Cowan, J A; Page, Richard C; Misra, Saurav


    Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.

  14. Interaction of organophosphorus pesticides with DNA nucleotides on a Boron-doped diamond electrode

    Energy Technology Data Exchange (ETDEWEB)

    Garbellini, Gustavo S.; Uliana, Carolina V.; Yamanaka, Hideko, E-mail: [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Bauru, SP (Brazil). Dept. de Quimica Analitica


    Diamond electrode was used to evaluate the interaction of the nucleotides guanosine monophosphate (GMP) and adenosine monophosphate (AMP) with the pesticides chlorpyrifos, methamidophos and monocrotophos. Changes were observed in the currents and peak potentials of the nucleotide voltammograms in the presence of the pesticides, with dependence on the pesticide concentration (from 5.0 Multiplication-Sign 10{sup -7} to 5.0 Multiplication-Sign 10{sup -5} mol L{sup -1}) and the interaction time (from 1 min to 4 h). This is probably due to binding of the pesticides to the nitrogenous bases present in the nucleotides, which could lead to problems in the DNA replication and biological functions of nucleotides. The pesticides showed stronger interaction with AMP than with GMP. Studies of the interaction of 50 Micro-Sign g mL{sup -1} DNA with the pesticides (from 30 min to 4 h and from 1.0 Multiplication-Sign 10{sup -6} to 6.0 Multiplication-Sign 10{sup -5} mol L{sup -1}) did not reveal any peaks relating to double helix opening or DNA unwinding. (author)

  15. Understanding specificity in metabolic pathways--structural biology of human nucleotide metabolism. (United States)

    Welin, Martin; Nordlund, Pär


    Interactions are the foundation of life at the molecular level. In the plethora of activities in the cell, the evolution of enzyme specificity requires the balancing of appropriate substrate affinity with a negative selection, in order to minimize interactions with other potential substrates in the cell. To understand the structural basis for enzyme specificity, the comparison of structural and biochemical data between enzymes within pathways using similar substrates and effectors is valuable. Nucleotide metabolism is one of the largest metabolic pathways in the human cell and is of outstanding therapeutic importance since it activates and catabolises nucleoside based anti-proliferative drugs and serves as a direct target for anti-proliferative drugs. In recent years the structural coverage of the enzymes involved in human nucleotide metabolism has been dramatically improved and is approaching completion. An important factor has been the contribution from the Structural Genomics Consortium (SGC) at Karolinska Institutet, which recently has solved 33 novel structures of enzymes and enzyme domains in human nucleotide metabolism pathways and homologs thereof. In this review we will discuss some of the principles for substrate specificity of enzymes in human nucleotide metabolism illustrated by a selected set of enzyme families where a detailed understanding of the structural determinants for specificity is now emerging.

  16. Variation in the nucleotide sequence of a prolamin gene family in wild rice. (United States)

    Barbier, P; Ishihama, A


    Variation in the DNA sequence of the 10 kDa prolamin gene family within the wild rice species Oryza rufipogon was probed using the direct sequencing of PCR-amplified genes. A comparison of the nucleotide and deduced amino-acid sequences of eight Asian strains of O. rufipogon and one strain of the related African species O. longistaminata is presented.

  17. Effects of 2'-O-methyl nucleotide substitution on EcoRI endonuclease cleavage activities.

    Directory of Open Access Journals (Sweden)

    Guojie Zhao

    Full Text Available To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl nucleotide (2'-OMeN to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2'-O-methyl nucleotide has a C3'-endo sugar pucker conformation different from the C2'-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2'-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2'-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2'-fluoro nucleic acid (2'-FNA and locked nucleic acid (LNA having similar C3'-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2'-endo to C3'-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2'-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.

  18. Nucleotide sequence and genomic organization of an ophiovirus associated with lettuce big-vein disease

    NARCIS (Netherlands)

    Wilk, van der F.; Dullemans, A.M.; Verbeek, M.; Heuvel, van den J.F.J.M.


    The complete nucleotide sequence of an ophiovirus associated with lettuce big-vein disease has been elucidated. The genome consisted of four RNA molecules of approximately 7ò8, 1ò7, 1ò5 and 1ò4 kb. Virus particles were shown to contain nearly equimolar amounts of RNA molecules of both polarities. Th

  19. LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E

    DEFF Research Database (Denmark)

    Jacobsen, Nana; Bentzen, Joan; Meldgaard, Michael;


    Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) ca...

  20. Complete Nucleotide Sequence of a Citrobacter freundii Plasmid Carrying KPC-2 in a Unique Genetic Environment (United States)

    Yao, Yancheng; Imirzalioglu, Can; Hain, Torsten; Kaase, Martin; Gatermann, Soeren; Exner, Martin; Mielke, Martin; Hauri, Anja; Dragneva, Yolanta; Bill, Rita; Wendt, Constanze; Wirtz, Angela; Chakraborty, Trinad


    The complete and annotated nucleotide sequence of a 54,036-bp plasmid harboring a blaKPC-2 gene that is clonally present in Citrobacter isolates from different species is presented. The plasmid belongs to incompatibility group N (IncN) and harbors the class A carbapenemase KPC-2 in a unique genetic environment. PMID:25395635

  1. Nucleotide sequence analysis of the lactococcal EPS plasmid pNZ4000

    NARCIS (Netherlands)

    Kranenburg, van R.; Kleerebezem, M.; Vos, de W.M.


    The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and thre

  2. A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization. (United States)

    Fu, Yuan; Lin, Hong-Yu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon


    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 typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

  3. Pain perception is altered by a nucleotide polymorphism in SCN9A.

    NARCIS (Netherlands)

    Reimann, F.; Cox, J.J.; Belfer, I.; Diatchenko, L.; Zaykin, D.V.; McHale, D.P.; Drenth, J.P.H.; Dai, F.; Wheeler, J.; Sanders, F.; Wood, L.; Wu, T.X.; Karppinen, J.; Nikolajsen, L.; Mannikko, M.; Max, M.B.; Kiselycznyk, C.; Poddar, M.; Morsche, R.H.M. te; Smith, S.; Gibson, D.; Kelempisioti, A.; Maixner, W.; Gribble, F.M.; Woods, C.G.


    The gene SCN9A is responsible for three human pain disorders. Nonsense mutations cause a complete absence of pain, whereas activating mutations cause severe episodic pain in paroxysmal extreme pain disorder and primary erythermalgia. This led us to investigate whether single nucleotide polymorphisms

  4. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications. (United States)


    ... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences... sequences are specifically excluded from this definition. Sequences with fewer than four specifically... acids are not intended to be embraced by this definition. Any amino acid sequence that contains...

  5. Development and characterization of 35 single nucleotide polymorphism markers for the brown alga Fucus vesiculosus

    NARCIS (Netherlands)

    Canovas, Fernando; Mota, Catarina; Ferreira-Costa, Joana; Serrao, Ester; Coyer, Jim; Olsen, Jeanine; Pearson, Gareth


    We characterized 35 single nucleotide polymorphism (SNP) markers for the brown alga Fucus vesiculosus. Based on existing Fucus Expressed Sequence Tag libraries for heat and desiccation-stressed tissue, SNPs were developed and confirmed by re-sequencing cDNA from a diverse panel of individuals. SNP l

  6. Twin Probes as a Novel Tool for the Detection of Single-Nucleotide Polymorphisms

    NARCIS (Netherlands)

    Ergen, Erhan; Weber, Markus; Jacob, Josemon; Herrmann, Andreas; Müllen, Klaus


    Single-nucleotide polymorphisms (SNPs) are the most common form of DNA sequence variation. There is a strong interest from both academy and industry to develop rapid, sensitive and cost effective methods for SNP detection. Here we report a novel structural concept for DNA detection based on fluoresc

  7. Affi-Gel Blue for nucleic acid removal and early enrichment of nucleotide binding proteins. (United States)

    Deutscher, Murray P


    Passage of an extract or supernatant fraction through a column of Affi-Gel Blue and batchwise elution can be a rapid and effective early procedure for removal of nucleic acid, concentration of the sample and purification of nucleotide binding proteins.

  8. Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly(iso-butylcyanoacrylate) nanocapsules. (United States)

    Hillaireau, H; Le Doan, T; Besnard, M; Chacun, H; Janin, J; Couvreur, P


    Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly(iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.

  9. Nucleotide excision repair in intact cells contrasts with high dual incision activity in vitro

    NARCIS (Netherlands)

    Jansen, J.; Olsen, A.K.; Wiger, R.; Naegeli, H.; Boer, de P.; Hoeven, van der F.; Holme, J.A.; Brunborg, G.; Mullenders, L.


    The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, gl

  10. The expected performance of single nucleotide polymorphism loci in paternity testing. (United States)

    Ayres, Karen L


    We discuss the utility of single nucleotide polymorphism loci for full trio and mother-unavailable paternity testing cases, in the presence of population substructure and relatedness of putative and actual fathers. We focus primarily on the expected number of loci required to gain specified probabilities of mismatches, and report the expected proportion of paternity indices greater than three threshold values for these loci.

  11. Should nucleotide sequence analyzing computer algorithms always extend homologies by extending homologies? (United States)

    Burnett, L; Basten, A; Hensley, W J


    Most computer algorithms used for comparing or aligning nucleotide sequences rely on the premise that the best way to extend a homology between the two sequences is to select a match rather than a mismatch. We have tested this assumption and found that it is not always valid.

  12. Nucleotide diversity patterns of local adaptation at drought-related candidate genes in wild tomatoes. (United States)

    Xia, Hui; Camus-Kulandaivelu, Létizia; Stephan, Wolfgang; Tellier, Aurélien; Zhang, Zhenwen


    We surveyed nucleotide diversity at two candidate genes LeNCED1 and pLC30-15, involved in an ABA (abscisic acid) signalling pathway, in two closely related tomato species Solanum peruvianum and Solanum chilense. Our six population samples (three for each species) cover a range of mesic to very dry habitats. The ABA pathway plays an important role in the plants' response to drought stress. LeNCED1 is an upstream gene involved in ABA biosynthesis, and pLC30-15 is a dehydrin gene positioned downstream in the pathway. The two genes show very different patterns of nucleotide variation. LeNCED1 exhibits very low nucleotide diversity relative to the eight neutral reference loci that were previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, pLC30-15 exhibits higher levels of nucleotide diversity and, in particular in S. chilense, higher genetic differentiation between populations than the reference loci, which is indicative of local adaptation. In the more drought-tolerant species S. chilense, one population (from Quicacha) shows a significant haplotype structure, which appears to be the result of positive (diversifying) selection.

  13. Understanding specificity in metabolic pathways-Structural biology of human nucleotide metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Welin, Martin [Structural Genomics Consortium, Karolinska Institutet, 17177 Stockholm (Sweden); Nordlund, Paer, E-mail: [Structural Genomics Consortium, Karolinska Institutet, 17177 Stockholm (Sweden); Division of Biophysics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm (Sweden)


    Interactions are the foundation of life at the molecular level. In the plethora of activities in the cell, the evolution of enzyme specificity requires the balancing of appropriate substrate affinity with a negative selection, in order to minimize interactions with other potential substrates in the cell. To understand the structural basis for enzyme specificity, the comparison of structural and biochemical data between enzymes within pathways using similar substrates and effectors is valuable. Nucleotide metabolism is one of the largest metabolic pathways in the human cell and is of outstanding therapeutic importance since it activates and catabolises nucleoside based anti-proliferative drugs and serves as a direct target for anti-proliferative drugs. In recent years the structural coverage of the enzymes involved in human nucleotide metabolism has been dramatically improved and is approaching completion. An important factor has been the contribution from the Structural Genomics Consortium (SGC) at Karolinska Institutet, which recently has solved 33 novel structures of enzymes and enzyme domains in human nucleotide metabolism pathways and homologs thereof. In this review we will discuss some of the principles for substrate specificity of enzymes in human nucleotide metabolism illustrated by a selected set of enzyme families where a detailed understanding of the structural determinants for specificity is now emerging.

  14. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA) (United States)

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

  15. Increasing the number of single nucleotide polymorphisms used in genomic evaluation of dairy cattle (United States)

    GeneSeek designed a new version of the GeneSeek Genomic Profiler HD BeadChip for Dairy Cattle, which had >77,000 single nucleotide polymorphisms (SNPs). A set of >140,000 SNPs was selected that included all SNPs on the existing GeneSeek chip, all SNPs used in U.S. national genomic evaluations, SNPs ...

  16. Nucleoside H-boranophosphonates: a new class of boron-containing nucleotide analogues. (United States)

    Higashida, Renpei; Oka, Natsuhisa; Kawanaka, Toshihide; Wada, Takeshi


    A study on the synthesis of nucleoside H-boranophosphonates, a new class of nucleotide analogues having a P-->BH(3) and a P-H group, via condensation of the corresponding nucleosides with H-boranophosphonate derivatives is described.

  17. Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops. (United States)

    Kent, Jessica L; McCann, Michael D; Phillips, Daniel; Panaro, Brandon L; Lim, Geoffrey F S; Serra, Martin J


    Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson-Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson-Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson-Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3' or 5' of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.

  18. Solubilization of a guanyl nucleotide-sensitive alpha/sub 1/ adrenergic receptor from liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Harris, S.I.; Moss, J.


    Rat liver membranes incubated with norepinephrine before solubilization with digitonin yielded a soluble hormone-receptor complex from which the release of tightly bound norepinephrine was facilitated by guanyl nucleotides. Binding of the alpha/sub 1/-adrenergic receptor antagonist, (/sup 3/H)-prazosin, to the soluble preparation was utilized as a gauge of guanyl nucleotide-induced release of receptor-bound agonist. The following potency series was obtained with regard to the ability of guanyl nucleotides to facilitate (/sup 3/H)-prazosin binding to the solubilized preparation: guanosine 5'-0-(3-thiotriphosphate)(K/sub 1/2/ = 2.5 nM), guanylyl-imidodiphosphate (K/sub 1/2/ = 10 nM), guanosine triphosphate (K/sub 1/2/ = 34 nM) and adenylyl-imidodiphosphate (K/sub 1/2/ > 1 mM). In the presence of guanylyl-imidodiphosphate (0.4 mM), the receptor population displayed monotonic binding parameters with a K/sub d/ for (/sup 3/H)-prazosin of 1.16 nM by Scatchard analysis. Competition curves against (/sup 3/H)-prazosin with the antagonists phentolamine and yohimbine revealed respective K/sub i/'s of .089 and 1.8; curves with the agonists norepinephrine and isoproterenol yielded respective K/sub i/'s of and 360 Competition curves performed in the absence of guanyl nucleotide were complex demonstrating an apparent increase in affinity for agonists and an apparent decrease in affinity for antagonists. These curve shifts are consistent with the conversion of receptor to and from the guanyl nucleotide-sensitive state as a function of competing ligand concentration.

  19. Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.). (United States)

    Katahira, Riko; Ashihara, Hiroshi


    As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --> nicotinamide mononucleotide --> nicotinamide riboside --> nicotinic acid riboside --> nicotinic acid mononucleotide --> nicotinic acid adenine dinucleotide --> NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.

  20. Colourimetric and spectroscopic discrimination between nucleotides and nucleosides using para-sulfonato-calix[4]arene capped silver nanoparticles. (United States)

    Tauran, Yannick; Grosso, Marie; Brioude, Arnaud; Kassab, Rima; Coleman, Anthony W


    The complexation of nucleosides and nucleotides by hybrid nanoparticles capped by para-sulfonato-calix[4]arene shows clear discrimination between purine and pyrimidine based molecules. For the pyrimidine nucleotides there is appearance of a new absorption band around 550 nm, and a colour change from yellow to orange red and pink.

  1. Functional Characterization of Single-Nucleotide Polymorphisms in the Human Undifferentiated Embryonic-Cell Transcription Factor 1 Gene

    NARCIS (Netherlands)

    Thummer, Rajkumar P.; Drenth-Diephuis, Loes J.; Carney, Karen E.; Eggen, Bart J. L.


    Single-nucleotide polymorphisms (SNPs) are single-nucleotide sequence variations between individuals. Two missense SNPs are present in the human undifferentiated embryonic-cell transcription factor 1 (UTF1) gene and their consequences for UTF1 function are investigated in this study. Expression of t

  2. Genome-wide divergence and linkage disequilibrium analyses for Capsicum baccatum revealed by genome-anchored single nucleotide polymorphisms (United States)

    Principal component analysis (PCA) with 36,621 polymorphic genome-anchored single nucleotide polymorphisms (SNPs) identified collectively for Capsicum annuum and Capsicum baccatum was used to show the distribution of these 2 important incompatible cultivated pepper species. Estimated mean nucleotide...

  3. IL-18 single nucleotide polymorphisms in hematologic malignancies with HLA matched sibling donor allogeneic hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the impact of interleukin-18(IL-18)single nucleotide polymorphisms on outcomes of hematologic malignancies with HLA-matched sibling donor hematopoietic stem cell transplantation(allo-HSCT).Methods Single-nucleotide polymorphisms in IL-18 promoter was detected by PCR-sequence-specific primer analysis(PCR-SSP)in 93 recipients and their HLA matched sibling donors.Hematopoietic reconstitution,

  4. Nucleotide variability and linkage disequilibrium patterns in the porcine MUC4 gene

    Directory of Open Access Journals (Sweden)

    Yang Ming


    Full Text Available Abstract Background MUC4 is a type of membrane anchored glycoprotein and serves as the major constituent of mucus that covers epithelial surfaces of many tissues such as trachea, colon and cervix. MUC4 plays important roles in the lubrication and protection of the surface epithelium, cell proliferation and differentiation, immune response, cell adhesion and cancer development. To gain insights into the evolution of the porcine MUC4 gene, we surveyed the nucleotide variability and linkage disequilibrium (LD within this gene in Chinese indigenous breeds and Western commercial breeds. Results A total of 53 SNPs covering the MUC4 gene were genotyped on 5 wild boars and 307 domestic pigs representing 11 Chinese breeds and 3 Western breeds. The nucleotide variability, haplotype phylogeny and LD extent of MUC4 were analyzed in these breeds. Both Chinese and Western breeds had considerable nucleotide diversity at the MUC4 locus. Western pig breeds like Duroc and Large White have comparable nucleotide diversity as many of Chinese breeds, thus artificial selection for lean pork production have not reduced the genetic variability of MUC4 in Western commercial breeds. Haplotype phylogeny analyses indicated that MUC4 had evolved divergently in Chinese and Western pigs. The dendrogram of genetic differentiation between breeds generally reflected demographic history and geographical distribution of these breeds. LD patterns were unexpectedly similar between Chinese and Western breeds, in which LD usually extended less than 20 kb. This is different from the presumed high LD extent (more than 100 kb in Western commercial breeds. The significant positive Tajima’D, and Fu and Li’s D statistics in a few Chinese and Western breeds implied that MUC4 might undergo balancing selection in domestic breeds. Nevertheless, we cautioned that the significant statistics could be upward biased by SNP ascertainment process. Conclusions Chinese and Western breeds have

  5. Dependence of the E.coli promoter strength and physical parameters upon the nucleotide sequence

    Institute of Scientific and Technical Information of China (English)



    The energy of interaction between complementary nucleotides in promoter sequences ofE. coli was calculated and visualized. The graphic method for presentation of energy properties of promoter sequences was elaborated on. Data obtained indicated that energy distribution through the length of promoter sequence results in picture with minima at -35, -8 and +7 regions corresponding to areas with elevated AT (adenine-thymine) content. The most important difference from the random sequences area is related to -8. Four promoter groups and their energy properties were revealed. The promoters with minimal and maximal energy of interaction between complementary nucleotides have low strengths, the strongest promoters correspond to promoter clusters characterized by intermediate energy values.

  6. Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins. (United States)

    Olszewska, Agata; Pohl, Radek; Brázdová, Marie; Fojta, Miroslav; Hocek, Michal


    Nucleotides, 2'-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53).

  7. An algorithm and program for finding sequence specific oligo-nucleotide probes for species identification

    Directory of Open Access Journals (Sweden)

    Tautz Diethard


    Full Text Available Abstract Background The identification of species or species groups with specific oligo-nucleotides as molecular signatures is becoming increasingly popular for bacterial samples. However, it shows also great promise for other small organisms that are taxonomically difficult to tract. Results We have devised here an algorithm that aims to find the optimal probes for any given set of sequences. The program requires only a crude alignment of these sequences as input and is optimized for performance to deal also with very large datasets. The algorithm is designed such that the position of mismatches in the probes influences the selection and makes provision of single nucleotide outloops. Program implementations are available for Linux and Windows.

  8. Twelve single nucleotide polymorphisms on chromosome 19q13.2-13.3

    DEFF Research Database (Denmark)

    Yin, Jiaoyang; Vogel, Ulla; Gerdes, Lars Ulrik;


    The genetic susceptibility to basal cell carcinoma (BCC) among Danish psoriatic patients was investigated in association studies with 12 single nucleotide polymorphisms on chromosome 19q13.2-3. The results show a significant association between BCC and the A-allele of a polymorphism in ERCCI exon4...... (Odds ratio 12;95% Confidence Interval 1.17-124; p(chi2, two-side) = 0.019) and to a lesser extent with XPD exon6 (p = 0.06). This is in accordance with recent studies of a different group of BCC cases (Rockenbauer et al. (in press) Carcinogenesis; Yin et al. (manuscript submitted for publication...... in nucleotide excision repair is of importance for the development of BCC....

  9. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites. (United States)

    Coutinho-Silva, Robson; Ojcius, David M


    Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.

  10. Thiolated pyrimidine nucleotides may interfere thiol groups concentrated at lipid rafts of HIV-1 infected cells. (United States)

    Kanizsai, Szilvia; Ongrádi, Joseph; Aradi, János; Nagy, Károly


    Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29. Selective cytotoxicity of thiolated pyrimidines on HIV-1 infected cells were observed. Results indicate that thiolated pyrimidine derivates may interfere with -SH (thiol) groups concentrated in lipid rafts of cell membrane and interacts HIV-1 infected (activated) cells resulting in a selective cytotoxicity of HIV-1 infected cells, and reducing HIV-1 entry.

  11. Ionic Liquids as Mobile Phase Additives for Separation of Nucleotides in High-Performance Liquid Chromatography

    Institute of Scientific and Technical Information of China (English)

    ZHANG,Wen-Zhu(张文珠); HE,Li-Jun(何丽君); LIU,Xia(刘霞); JIANG,Sheng-Xiang(蒋生祥)


    Ionic liquids are a type of salts that are liquid at low temperature (< 100 ℃). Because of their some special properties, they have been widely used as new "green solvents" for many chemical reactions and liquid-liquid extraction in the past several years. In this paper, a new method for the separation of nucleotides is developed and the essential feature of the method is that 1-alkyl-3-methylimidazolium salts are used as mobile phase additives, resulting in a baseline separation of nucleotides without need of gradient elution and need of organic solvent addition as currently used in RP-HPLC. This study shows the potential application of ionic liquids as mobile phase additives in reversed-phase liquid chromatography.

  12. Machine Learning Techniques for Single Nucleotide Polymorphism—Disease Classification Models in Schizophrenia

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    Cristian R. Munteanu


    Full Text Available Single nucleotide polymorphisms (SNPs can be used as inputs in disease computational studies such as pattern searching and classification models. Schizophrenia is an example of a complex disease with an important social impact. The multiple causes of this disease create the need of new genetic or proteomic patterns that can diagnose patients using biological information. This work presents a computational study of disease machine learning classification models using only single nucleotide polymorphisms at the HTR2A and DRD3 genes from Galician (Northwest Spain schizophrenic patients. These classification models establish for the first time, to the best knowledge of the authors, a relationship between the sequence of the nucleic acid molecule and schizophrenia (Quantitative Genotype – Disease Relationships that can automatically recognize schizophrenia DNA sequences and correctly classify between 78.3–93.8% of schizophrenia subjects when using datasets which include simulated negative subjects and a linear artificial neural network.

  13. Effects of preservation methods on amino acids and 5'-nucleotides of Agaricus bisporus mushrooms. (United States)

    Liu, Ying; Huang, Fan; Yang, Hong; Ibrahim, S A; Wang, Yan-Feng; Huang, Wen


    In this study, the proximate composition, free amino acids content and 5'-nucleotides in frozen, canned and salted Agaricus bisporus (A. bisporus) were investigated. We found that the three kinds of A. bisporus products were good sources of protein, with amount varying in the ranges of 16.54-24.35g/100g (dry weight). Freezing, canning and salting process, followed by 6months of storage led to a significant reduction in free amino acids, especially tyrosine, alanine, glutamine and cysteine. There were medium levels of MSG-like amino acids in frozen A. bisporus and canned A. bisporus, and low levels of MSG-like amino acids in salted A. bisporus. The mount of flavor 5'-nucleotides in frozen A. bisporus was higher than that of canned and salted A. bisporus. The present study thus suggests that freezing is beneficial for the preservation of A. bisporus.

  14. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a. (United States)

    Vanderslice, P; Copeland, W C; Robertus, J D


    Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

  15. A genetic variation map for chicken with 2.8 million single nucleotide polymorphisms

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    Wong, G K; Hillier, L; Brandstrom, M; Croojmans, R; Ovcharenko, I; Gordon, L; Stubbs, L; Lucas, S; Glavina, T; Kaiser, P; Gunnarsson, U; Webber, C; Overton, I


    We describe a genetic variation map for the chicken genome containing 2.8 million single nucleotide polymorphisms (SNPs), based on a comparison of the sequences of 3 domestic chickens (broiler, layer, Silkie) to their wild ancestor Red Jungle Fowl (RJF). Subsequent experiments indicate that at least 90% are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about 5 SNP/kb for almost every possible comparison between RJF and domestic lines, between two different domestic lines, and within domestic lines--contrary to the idea that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated prior to domestication, and there is little to no evidence of selective sweeps for adaptive alleles on length scales of greater than 100 kb.

  16. Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    赛吉庆; 康良仪; 黄忠; 史春霖; 田波; 谢友菊


    The 3’-terminal 1 279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined. This sequence contains an open reading frame of 1023 nudeotides and a 3’ -non-coding region of 256 nucleotides. The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (Nib). The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35400. The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses. The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E. coli cells. The fusion polypeptide positively reacted in Western blot with an antiserum prepared against the native viral CP.

  17. Nucleotide polymorphism in the drought responsive gene Asr2 in wild populations of tomato. (United States)

    Giombini, Mariano I; Frankel, Nicolás; Iusem, Norberto D; Hasson, Esteban


    The Asr gene family (named after abscicic acid [ABA], stress, ripening), exclusively present in plant genomes, is involved in transcriptional regulation. Its members are up-regulated in roots and leaves of water- or salt-stressed plants. In previous work, evidence of adaptive evolution (as inferred from synonymous and nonsynonymous divergence rates) has been reported for Asr2 in Solanum chilense and S. arcanum, two species dwelling in habitats with different precipitation regimes. In this paper we investigate patterns of intraspecific nucleotide variation in Asr2 and the unlinked locus CT114 in S. chilense and S. arcanum. The extent of nucleotide diversity in Asr2 differed between species in more than one order of magnitude. In both species we detected evidence of non-neutral evolution, which may be ascribed to different selective regimes, potentially associated to unique climatic features, or, alternatively, to demographic events. The results are discussed in the light of demographic and selective hypotheses.

  18. The nucleotide sequence of 4.5S ribosomal RNA from tobacco chloroplasts.


    Takaiwa, F; Sugiura, M


    The nucleotide sequence of tobacco chloroplast 4.5S ribosomal RNA has been determined to be: OHG-A-A-G-G-U-C-A-C-G-G-C-G-A-G-A-C-G-A-G-C-C-G-U-U-U-A-U-C-A-U-U-A-C-G-A-U-A-G-G-U-G-U-C-A-A-G-U-G-G-A-A-G-U-G-C-A-G-U-G-A-U-G-U-A-U-G-C-(G-A)-C-U-G-A-G-G-C-A-U-C-C-U-A-A-C-A-G-A-C-C-G-G-U-A-G-A-C-U-U-G-A-A-COH. The 4.5S RNA is 103 nucleotides long and its 5'-terminus is not phosphorylated.

  19. Nucleotide sequencing and serologic analysis of Cache Valley virus isolates from the Yucatan Peninsula of Mexico. (United States)

    Blitvich, Bradley J; Loroño-Pino, Maria A; Garcia-Rejon, Julian E; Farfan-Ale, Jose A; Dorman, Karin S


    Nucleotide sequencing was performed on part of the medium and large genome segments of 17 Cache Valley virus (CVV) isolates from the Yucatan Peninsula of Mexico. Alignment of these sequences to all other sequences in the Genbank database revealed that they have greatest nucleotide identity (97-98 %) with the equivalent regions of Tlacotalpan virus (TLAV), which is considered to be a variety of CVV. Next, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice that had been inoculated with a representative isolate from the Yucatan Peninsula (CVV-478) or the prototype TLAV isolate (61-D-240). The PRNT titers exhibited a twofold difference in one direction and no difference in the other direction suggesting that CVV-478 and 61-D-240 belong to the same CVV subtype. In conclusion, we demonstrate that the CVV isolates from the Yucatan Peninsula of Mexico are genetically and antigenically similar to the prototype TLAV isolate.

  20. Efficient single nucleotide polymorphism discovery in laboratory rat strains using wild rat-derived SNP candidates


    Hedrich Hans J; Wedekind Dirk; Zeegers Dimphy; Guryev Victor; Smits Bart MG; Cuppen Edwin


    Abstract Background The laboratory rat (Rattus norvegicus) is an important model for studying many aspects of human health and disease. Detailed knowledge on genetic variation between strains is important from a biomedical, particularly pharmacogenetic point of view and useful for marker selection for genetic cloning and association studies. Results We show that Single Nucleotide Polymorphisms (SNPs) in commonly used rat strains are surprisingly well represented in wild rat isolates. Shotgun ...