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Sample records for ef-tu mutants designed

  1. Reducing ppGpp level rescues an extreme growth defect caused by mutant EF-Tu.

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    Jessica M Bergman

    Full Text Available Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.

  2. Structural studies of EF-Tu complexes

    DEFF Research Database (Denmark)

    Johansen, Jesper Sanderhoff

    2011-01-01

    Protein synthesis is a process vital to all living organisms. Protein synthesis occurs on large ribonucleoprotein complexes called ribosomes. Amino acids (aa) are brought to the ribosome as part of a ternary complex consisting of elongation factor EF-Tu, GTP and aminoacyl-tRNA (aa-tRNA). The GTP...... elongation factor, EF-Ts, catalyse the dissociation of GDP from EF-Tu which in turn allows EF-Tu to bind another GTP molecule. The resulting EF-Tu:GTP complex is then cable of forming a ternary complex with a new aa-tRNA which can then associate with the ribosome. During my time as a Ph. D. student I have...... proteins and 22 tRNAs and 2 rRNAs. The mammalian mitochondria have their own specialised translational system for maintaining the synthesis of the 13 proteins. The mammalian mitochondrial protein synthesis resembles the prokaryotic system more than the cytosolic system from eukaryotes. Some of the t...

  3. Cloning and Characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa

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    Stephanie O. Palmer

    2013-01-01

    Full Text Available We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be = 33 μM, = 0.003 s−1, and the specificity constant was  s−1 μM−1. In the presence of EF-Ts, these values were shifted to = 2 μM, = 0.005 s−1, and the specificity constant was  s−1 μM−1. The equilibrium dissociation constants governing the binding of EF-Tu to GDP ( were 30–75 nM and to GTP ( were 125–200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U-dependent binding of Phe-tRNAPhe at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

  4. Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.

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    Palmer, Stephanie O; Rangel, Edna Y; Montalvo, Alberto E; Tran, Alexis T; Ferguson, Kate C; Bullard, James M

    2013-01-01

    We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 μM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) μM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 μM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) μM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

  5. Nucleotide and aminoacyl-tRNA specificity of the mammalian mitochondrial elongation factor EF-Tu.Ts complex.

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    Woriax, V L; Spremulli, G H; Spremulli, L L

    1996-06-03

    The bovine mitochondrial elongation factor Tu.Ts complex (EF-Tu.Tsmt) promotes the binding of aminoacyl-tRNA to ribosomes. In the presence of GTP, this complex functions catalytically. Both dGTP and ddGTP can replace GTP although about 4-fold higher concentrations are required. ATP, CTP and UTP are not active. ITP can replace GTP when used at 10- to 20-fold higher concentrations. The catalytic use of EF-Tu.Tsmt is inhibited by GDP but not by GMP. XDP also inhibits although about 20-fold higher concentrations are required. EF-Tu.Tsmt will promote the binding of Phe-tRNA to either Escherichia coli or mitochondrial ribosomes. Unlike E. coli EF-Tu, EF-Tu.Tsmt will promote the binding of AcPhe-tRNA to ribosomes about 25% as efficiently as Phe-tRNA. EF-Tu.Tsmt is active in catalyzing the binding of E. coli Met-tRNAmmet to ribosomes. EF-Tu.Tsmt has about 30% as much activity with E. coli Met-tRNAimet but has essentially no activity with E. coli fMet-tRNAimet. Neither yeast Met-tRNAimet nor fMet-tRNAimet is recognized by bovine EF-Tu.Tsmt.

  6. Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits

    DEFF Research Database (Denmark)

    Blumenthal, T; Young, R A; Brown, S

    1976-01-01

    Qbeta replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein S1 and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The affinity of the two subcomplexes...... alters its quaternary structure: the EF-Tu-Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA. This conformational change is not influenced by ionic strength. The addition of Qbeta RNA to the enzyme, does not result in the release...... of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP...

  7. Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

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    Takeshita, Daijiro; Tomita, Kozo

    2010-09-07

    Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

  8. The effects of EF-Ts and bismuth on EF-Tu in Helicobacter pylori: implications for an elegant timing for the introduction of EF-Ts in the elongation and EF-Tu as a potential drug target.

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    Wang, Dongxian; Luo, Benping; Shan, Weiran; Hao, Mingcong; Sun, Xuesong; Ge, Ruiguang

    2013-06-01

    Helicobacter pylori is a common human pathogen responsible for various gastric diseases. Bismuth can effectively inhibit the growth of this bacterium and is commonly recommended for the treatment of the related diseases. Translation elongation factors EF-Tu and EF-Ts are two important components of the protein translation system. EF-Ts has inhibitory effects on the GTPase activity of EF-Tu and enhances GDP release, a hint that careful timing for the introduction of EF-Ts in the elongation should be accomplished to prevent the complete inhibition of the elongation process. Bismuth inhibits the chaperone activity of EF-Tu, and has opposite effects on the elongation activity: inhibitory effects on the intrinsic GTPase activity and stimulation of GDP release. The present work deepens our understanding of the bacterial elongation process as mediated by EF-Tu and EF-Ts and extends our knowledge about the inhibitory effects of bismuth-based drugs against Helicobacter pylori.

  9. A neutron scattering study of the ternary complex EF-Tu.GTP-valyl-tRNAVal1A

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    Österberg, R.; Elias, P.; Kjems, Jørgen

    1986-01-01

    The complex formation between elongation factor Tu (EF-Tu), GTP, and valyl-tRNAVal1A has been investigated in a hepes buffer of "pH" 7.4 and 0.2 M ionic strength using the small-angle neutron scattering method at concentrations of D2O where EF-Tu (42% D2O) and tRNA (71% D2O) are successively...

  10. Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues.

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    Sato, Aya; Suematsu, Takuma; Aihara, Koh-Ki; Kita, Kiyoshi; Suzuki, Tsutomu; Watanabe, Kimitsuna; Ohtsuki, Takashi; Watanabe, Yoh-Ichi

    2017-03-07

    Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegansC. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  11. Arabidopsis EF-Tu receptor enhances bacterial disease resistance in transgenic wheat.

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    Schoonbeek, Henk-Jan; Wang, Hsi-Hua; Stefanato, Francesca L; Craze, Melanie; Bowden, Sarah; Wallington, Emma; Zipfel, Cyril; Ridout, Christopher J

    2015-04-01

    Perception of pathogen (or microbe)-associated molecular patterns (PAMPs/MAMPs) by pattern recognition receptors (PRRs) is a key component of plant innate immunity. The Arabidopsis PRR EF-Tu receptor (EFR) recognizes the bacterial PAMP elongation factor Tu (EF-Tu) and its derived peptide elf18. Previous work revealed that transgenic expression of AtEFR in Solanaceae confers elf18 responsiveness and broad-spectrum bacterial disease resistance. In this study, we developed a set of bioassays to study the activation of PAMP-triggered immunity (PTI) in wheat. We generated transgenic wheat (Triticum aestivum) plants expressing AtEFR driven by the constitutive rice actin promoter and tested their response to elf18. We show that transgenic expression of AtEFR in wheat confers recognition of elf18, as measured by the induction of immune marker genes and callose deposition. When challenged with the cereal bacterial pathogen Pseudomonas syringae pv. oryzae, transgenic EFR wheat lines had reduced lesion size and bacterial multiplication. These results demonstrate that AtEFR can be transferred successfully from dicot to monocot species, further revealing that immune signalling pathways are conserved across these distant phyla. As novel PRRs are identified, their transfer between plant families represents a useful strategy for enhancing resistance to pathogens in crops. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  12. Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodiumfalciparum plastid.

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    Biswas, Subir; Lim, Erin E; Gupta, Ankit; Saqib, Uzma; Mir, Snober S; Siddiqi, Mohammad Imran; Ralph, Stuart A; Habib, Saman

    2011-03-01

    Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome. Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.

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

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

    2013-09-01

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

  14. How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome

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    Noel, Jeffrey K.; Whitford, Paul C.

    2016-10-01

    It has long been recognized that the thermodynamics of mRNA-tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. To rationalize this apparent inconsistency, Hopfield proposed that the ribosome may improve accuracy by utilizing a multi-step kinetic proofreading mechanism. While biochemical, structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical-chemical properties of the ribosome enable proofreading. To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. We find that the steric composition of EF-Tu can reduce the free-energy barrier associated with the first step of accommodation: elbow accommodation. We interpret this effect within an extended kinetic model of accommodation and show how EF-Tu can contribute to efficient and accurate proofreading.

  15. The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange*

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    De Laurentiis, Evelina Ines; Mercier, Evan; Wieden, Hans-Joachim

    2016-01-01

    Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli. Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts. PMID:27624934

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

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

  17. Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: segregation pattern, expression and effects of the transgene.

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    Fu, Jianming; Ristic, Zoran

    2010-06-01

    We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation following exposure to heat stress (18 h at 45 degrees C) [Fu et al. in Plant Mol Biol 68:277-288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45 degrees C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.

  18. Robust mutant strain design by pessimistic optimization.

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    Apaydin, Meltem; Xu, Liang; Zeng, Bo; Qian, Xiaoning

    2017-10-03

    proposed pessimistic formulations yield higher chemical production rates than those by the optimistic formulations. Moreover, with higher uncertainty levels, both cellular models under pessimistic approaches produce the same mutant strains. In this paper, we propose a new pessimistic optimization framework for mutant strain design. Our pessimistic strain optimization methods produce more robust solutions regardless of the inner-level mutant survival models, which is desired as the models for cell survival are often approximate to real-world systems. Such robust and reliable knockout strategies obtained by the pessimistic formulations would provide confidence for in-vivo experimental design of microbial mutants of interest.

  19. Making designer mutants in model organisms

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    Peng, Ying; Clark, Karl J.; Campbell, Jarryd M.; Panetta, Magdalena R.; Guo, Yi; Ekker, Stephen C.

    2014-01-01

    Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms. PMID:25336735

  20. Rational design of an enzyme mutant for anti-cocaine therapeutics

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    Zheng, Fang; Zhan, Chang-Guo

    2008-09-01

    (-)-Cocaine is a widely abused drug and there is no available anti-cocaine therapeutic. The disastrous medical and social consequences of cocaine addiction have made the development of an effective pharmacological treatment a high priority. An ideal anti-cocaine medication would be to accelerate (-)-cocaine metabolism producing biologically inactive metabolites. The main metabolic pathway of cocaine in body is the hydrolysis at its benzoyl ester group. Reviewed in this article is the state-of-the-art computational design of high-activity mutants of human butyrylcholinesterase (BChE) against (-)-cocaine. The computational design of BChE mutants have been based on not only the structure of the enzyme, but also the detailed catalytic mechanisms for BChE-catalyzed hydrolysis of (-)-cocaine and (+)-cocaine. Computational studies of the detailed catalytic mechanisms and the structure-and-mechanism-based computational design have been carried out through the combined use of a variety of state-of-the-art techniques of molecular modeling. By using the computational insights into the catalytic mechanisms, a recently developed unique computational design strategy based on the simulation of the rate-determining transition state has been employed to design high-activity mutants of human BChE for hydrolysis of (-)-cocaine, leading to the exciting discovery of BChE mutants with a considerably improved catalytic efficiency against (-)-cocaine. One of the discovered BChE mutants (i.e., A199S/S287G/A328W/Y332G) has a ˜456-fold improved catalytic efficiency against (-)-cocaine. The encouraging outcome of the computational design and discovery effort demonstrates that the unique computational design approach based on the transition-state simulation is promising for rational enzyme redesign and drug discovery.

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

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    Satya Brata Routh

    2016-05-01

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

  2. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Kožíšek, Milan

    2014-01-01

    to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence......-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein....

  3. Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants.

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    Havenith, Heide; Kern, Karolin; Rautenberger, Paul; Spiegel, Holger; Szardenings, Michael; Ueberham, Elke; Lehmann, Jörg; Buntru, Matthias; Vogel, Simon; Treudler, Regina; Fischer, Rainer; Schillberg, Stefan

    2017-02-01

    Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Structure of the E. coli ribosome-EF-Tu complex at EM.

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    Fischer, Niels; Neumann, Piotr; Konevega, Andrey L; Bock, Lars V; Ficner, Ralf; Rodnina, Marina V; Stark, Holger

    2015-04-23

    Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8 Å), but provides more detailed information (2.65 Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

  5. Rationally Designed PI3Kα Mutants to Mimic ATR and Their Use to Understand Binding Specificity of ATR Inhibitors.

    Science.gov (United States)

    Lu, Yipin; Knapp, Mark; Crawford, Kenneth; Warne, Robert; Elling, Robert; Yan, Kelly; Doyle, Michael; Pardee, Gwynn; Zhang, Li; Ma, Sylvia; Mamo, Mulugeta; Ornelas, Elizabeth; Pan, Yue; Bussiere, Dirksen; Jansen, Johanna; Zaror, Isabel; Lai, Albert; Barsanti, Paul; Sim, Janet

    2017-06-02

    ATR, a protein kinase in the PIKK family, plays a critical role in the cell DNA-damage response and is an attractive anticancer drug target. Several potent and selective inhibitors of ATR have been reported showing significant antitumor efficacy, with most advanced ones entering clinical trials. However, due to the absence of an experimental ATR structure, the determinants contributing to ATR inhibitors' potency and specificity are not well understood. Here we present the mutations in the ATP-binding site of PI3Kα to progressively transform the pocket to mimic that of ATR. The generated PI3Kα mutants exhibit significantly improved affinity for selective ATR inhibitors in multiple chemical classes. Furthermore, we obtained the X-ray structures of the PI3Kα mutants in complex with the ATR inhibitors. The crystal structures together with the analysis on the inhibitor affinity profile elucidate the roles of individual amino acid residues in the binding of ATR inhibitors, offering key insights for the binding mechanism and revealing the structure features important for the specificity of ATR inhibitors. The ability to obtain structural and binding data for these PI3Kα mutants, together with their ATR-like inhibitor binding profiles, makes these chimeric PI3Kα proteins valuable model systems for structure-based inhibitor design. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  6. Computational design of novel inhibitors to overcome weed resistance associated with acetohydroxyacid synthase (AHAS) P197L mutant.

    Science.gov (United States)

    Qu, Ren-Yu; Yang, Jing-Fang; Liu, Yu-Chao; Chen, Qiong; Hao, Ge-Fei; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

    2017-07-01

    Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. With the long-term and wide application of AHAS inhibitors, weed resistance is becoming a global problem, which leads to an urgent demand for novel inhibitors to antagonize both wild-type and resistant AHAS. Pyrimidinyl salicylic acid derivatives, as one of the main classes of commercial AHAS herbicides, show potential anti-resistant bioactivity to wild-type and P197L mutant. In current work, a series of novel 2-benzoyloxy-6-pyrimidinyl salicylic acid derivatives were designed through fragment-based drug discovery. Fortunately, the newly synthesized compounds showed good inhibitory activity against both wild-type and P197L mutant. Some compounds not only had a lower resistance factor value but also showed excellent inhibitory activity against wild-type AHAS and P197L mutant. Furthermore, greenhouse experiments showed compound 11m displayed almost 100% inhibition against both wild-type and high-resistant Descurainia sophia at a dosage of 150 g a.i. ha-1 . The present work indicated that the 2-benzoyloxy-6-pyrimidinyl salicylic acid motif was well worth further optimization. Also, compound 11m could be used as a potential anti-resistant AHAS herbicide, which requires further research. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  7. Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

    Science.gov (United States)

    Shrestha, Suresh; Salins, Lyndon L E.; Mark Ensor, C.; Daunert, Sylvia

    2002-01-01

    Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the

  8. Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides

    Directory of Open Access Journals (Sweden)

    Bogumil Jacek Karas

    2014-07-01

    Full Text Available With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in

  9. Bottom-up design of small molecules that stimulate exon 10 skipping in mutant MAPT pre-mRNA.

    Science.gov (United States)

    Luo, Yiling; Disney, Matthew D

    2014-09-22

    One challenge in chemical biology is to develop small molecules that control cellular protein content. The amount and identity of proteins are influenced by the RNAs that encode them; thus, protein content in a cell could be affected by targeting mRNA. However, RNA has been traditionally difficult to target with small molecules. In this report, we describe controlling the protein products of the mutated microtubule-associated protein tau (MAPT) mature mRNA with a small molecule. MAPT mutations in exon 10 are associated with inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), an incurable disease that is directly caused by increased inclusion of exon 10 in MAPT mRNA. Recent studies have shown that mutations within a hairpin at the MAPT exon 10-intron junction decrease the thermodynamic stability of the RNA, increasing binding to U1 snRNP and thus exon 10 inclusion. Therefore, we designed small molecules that bind and stabilize a mutant MAPT by using Inforna, a computational approach based on information about RNA-small-molecule interactions. The optimal compound selectively bound the mutant MAPT hairpin and thermodynamically stabilized its folding, facilitating exon 10 exclusion. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. ATM mutants

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. ATM mutants. ATM (Ataxia Telangiectasia Mutated). AT2BE and AT5B1 cells – fibroblast cell lines from Ataxia telangiectasia patients. Deletion mutants expressing truncated ATM protein which is inactive. Have been used in studies looking at the role of ATM in DNA damage ...

  11. Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

    Directory of Open Access Journals (Sweden)

    Roland Züst

    Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

  12. Design of Factor XIII V34X Activation Peptides to Control Ability to Interact With Thrombin Mutants

    Science.gov (United States)

    Jadhav, Madhavi A.; Lucas, R. Cory; Goldsberry, Whitney N.; Maurer, Muriel C.

    2011-01-01

    Thrombin helps to activate Factor XIII (FXIII) by hydrolyzing the R37-G38 peptide bond. The resultant transglutaminase introduces cross-links into the fibrin clot. With the development of therapeutic coagulation factors, there is a need to better understand interactions involving FXIII. Such knowledge will help predict ability to activate FXIII and thus ability to promote/hinder the generation of transglutaminase activity. Kinetic parameters have been determined for a series of thrombin species hydrolyzing the FXIII (28′41) V34X activation peptides (V34, V34L, V34F, and V34P). The V34P substitution introduces PAR4 character into the FXIII, and the V34F exhibits important similarities to the cardioprotective V34L. FXIII activation peptides containing V34, V34L, or V34P could each be accommodated by alanine mutants of thrombin lacking either the W60d or Y60a residue in the 60-insertion loop. By contrast, FXIII V34F AP could be cleaved by thrombin W60dA but not by Y60aA. FXIII V34P is highly reliant on the thrombin W215 platform for its strong substrate properties whereas FXIII V34F AP becomes the first segment that can maintain its Km upon loss of the critical thrombin W215 residue. Interestingly, FXIII V34F AP could also be readily accommodated by thrombin L99A and E217A. Hydrolysis of FXIII V34F AP by thrombin W217A/E217A (WE) was similar to that of FXIII V34L AP whereas WE could not effectively cleave FXIII V34P AP. FXIII V34F and V34P AP show promise for designing FXIII activation systems that are either tolerant of or greatly hindered by the presence of anticoagulant thrombins. PMID:21798378

  13. Rational design of viscosity reducing mutants of a monoclonal antibody: hydrophobic versus electrostatic inter-molecular interactions.

    Science.gov (United States)

    Nichols, Pilarin; Li, Li; Kumar, Sandeep; Buck, Patrick M; Singh, Satish K; Goswami, Sumit; Balthazor, Bryan; Conley, Tami R; Sek, David; Allen, Martin J

    2015-01-01

    High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region. The disruption of an APR that lies at the interface of light and heavy chain variable domains, VH and VL, via L45K mutation destabilized MAB 1 and abolished antigen binding. However, mutation at the preceding residue (V44K), which also lies in the same APR, increased apparent solubility and reduced viscosity of MAB 1 without sacrificing antigen binding or thermal stability. Neutralizing the negatively charged surface patch (E59Y) also increased apparent solubility and reduced viscosity of MAB 1, but charge reversal at the same position (E59K/R) caused destabilization, decreased solubility and led to difficulties in sample manipulation that precluded their viscosity measurements at high concentrations. Both V44K and E59Y mutations showed similar increase in apparent solubility. However, the viscosity profile of E59Y was considerably better than that of the V44K, providing evidence that inter-molecular interactions in MAB 1 are electrostatically driven. In conclusion, neutralizing negatively charged surface patches may be more beneficial toward reducing viscosity of highly concentrated antibody solutions than charge reversal or aggregation prone motif disruption.

  14. Design and characterization of genetically engineered zebrafish aquaporin-3 mutants highly permeable to the cryoprotectant ethylene glycol

    Directory of Open Access Journals (Sweden)

    Lubzens Esther

    2011-04-01

    Full Text Available Abstract Background Increasing cell membrane permeability to water and cryoprotectants is critical for the successful cryopreservation of cells with large volumes. Artificial expression of water-selective aquaporins or aquaglyceroporins (GLPs, such as mammalian aquaporin-3 (AQP3, enhances cell permeability to water and cryoprotectants, but it is known that AQP3-mediated water and solute permeation is limited and pH dependent. To exploit further the possibilities of using aquaporins in cryobiology, we investigated the functional properties of zebrafish (Danio rerio GLPs. Results Water, glycerol, propylene glycol and ethylene glycol permeability of zebrafish Aqp3a, -3b, -7, -9a, -9b, -10a and -10b, and human AQP3, was examined. Expression in Xenopus laevis oocytes indicated that the permeability of DrAqp3a and -3b to ethylene glycol was higher than for glycerol or propylene glycol under isotonic conditions, unlike other zebrafish GLPs and human AQP3, which were more permeable to glycerol. In addition, dose-response experiments and radiolabeled ethylene glycol uptake assays suggested that oocytes expressing DrAqp3b were permeated by this cryoprotectant more efficiently than those expressing AQP3. Water and ethylene glycol transport through DrAqp3a and -3b were, however, highest at pH 8.5 and completely abolished at pH 6.0. Point mutations in the DrAqp3b amino acid sequence rendered two constructs, DrAqp3b-T85A showing higher water and ethylene glycol permeability at neutral and alkaline pH, and DrAqp3b-H53A/G54H/T85A, no longer inhibited at acidic pH but less permeable than the wild type. Finally, calculation of permeability coefficients for ethylene glycol under concentration gradients confirmed that the two DrAqp3b mutants were more permeable than wild-type DrAqp3b and/or AQP3 at neutral pH, resulting in a 2.6- to 4-fold increase in the oocyte intracellular concentration of ethylene glycol. Conclusion By single or triple point mutations in the Dr

  15. Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

    2014-01-01

    Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing...... acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects. © 2014 Springer-Verlag Berlin Heidelberg....

  16. Identification of possible siRNA molecules for TDP43 mutants causing amyotrophic lateral sclerosis: In silico design and molecular dynamics study.

    Science.gov (United States)

    Bhandare, Vishwambhar Vishnu; Ramaswamy, Amutha

    2016-04-01

    The DNA binding protein, TDP43 is a major protein involved in amyotrophic lateral sclerosis and other neurological disorders such as frontotemporal dementia, Alzheimer disease, etc. In the present study, we have designed possible siRNAs for the glycine rich region of tardbp mutants causing ALS disorder based on a systematic theoretical approach including (i) identification of respective codons for all mutants (reported at the protein level) based on both minimum free energy and probabilistic approaches, (ii) rational design of siRNA, (iii) secondary structure analysis for the target accessibility of siRNA, (iii) determination of the ability of siRNA to interact with mRNA and the formation/stability of duplex via molecular dynamics study for a period of 15ns and (iv) characterization of mRNA-siRNA duplex stability based on thermo-physical analysis. The stable GC-rich siRNA expressed strong binding affinity towards mRNA and forms stable duplex in A-form. The linear dependence between the thermo-physical parameters such as Tm, GC content and binding free energy revealed the ability of the identified siRNAs to interact with mRNA in comparable to that of the experimentally reported siRNAs. Hence, this present study proposes few siRNAs as the possible gene silencing agents in RNAi therapy based on the in silico approach. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Design and Synthesis of Bis-Biotin-Containing Reagents for Applications Utilizing Monoclonal Antibody-Based Pretargeting Systems with Streptavidin Mutants

    Science.gov (United States)

    Wilbur, D. Scott; Park, Steven I.; Chyan, Ming-Kuan; Wan, Feng; Hamlin, Donald K.; Shenoi, Jaideep; Lin, Yukang; Wilbur, Shani M.; Buchegger, Franz; Pantelias, Anastasia; Pagel, John M.; Press, Oliver W.

    2010-01-01

    Previous studies have shown that pretargeting protocols, using cancer-targeting fusion proteins, composed of 4 anti-CD20 single chain Fv (scFv) fragments and streptavidin (scFv4-SAv), followed by a biotinylated dendrimeric N-acetyl-galactosamine blood clearing agent (CA), 1, then a radiolabeled DOTA-biotin derivative (a mono-biotin), 3a, can provide effective therapy for lymphoma xenografts in mouse models. A shortcoming in this pretargeting system is that endogenous biotin may affect its efficacy in patients. To circumvent this potential problem, we investigated a pretargeting system that employs anti-CD20 scFv4-SAv mutant fusion proteins with radioiodinated bis-biotin derivatives. With that combination of reagents good localization of the radiolabel to lymphoma tumor xenografts was obtained in the presence of endogenous biotin. However, the blood clearance reagents employed in the studies were ineffective, resulting in abnormally high levels of radioactivity in other tissues. Thus, in the present investigation a bis-biotin-tri-galactose blood clearance reagent, 2, was designed, synthesized and evaluated in vivo. Additionally, another DOTA-biotin derivative (a bis-biotin), 4a, was designed and synthesized, such that radiometals (e.g. 111In, 90Y, 177Lu) could be used in the pretargeting protocols employing scFv4-SAv mutant fusion proteins. Studies in mice demonstrated that the CA 2 was more effective than CA 1 at removing [125I]scFv4-SAv-S45A mutant fusion proteins from blood. Another in vivo study compared tumor targeting and normal tissue concentrations of the new reagents (2 & [111In]4b) with standard reagents (1 and [111In]3b) used in pretargeting protocols. The study showed that lymphoma xenografts could be targeted in the presence of endogenous biotin when anti-CD20 fusion proteins containing SAv mutants (scFv4-SAv-S45A or scFv4-SAv-Y43A) were employed in combination with CA 2 and [111In]4b. Importantly, normal tissue concentrations of [111In]4b were similar

  18. Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

    Directory of Open Access Journals (Sweden)

    Liang Xu

    2017-09-01

    Full Text Available Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26, a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action

  19. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; CLARK, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  20. Clearance of mutant huntingtin.

    Science.gov (United States)

    Li, Xiao-Jiang; Li, He; Li, Shihua

    2010-07-01

    Mutant huntingtin (htt) carries an expanded polyglutamine (polyQ) repeat (> 36 glutamines) in its N-terminal region, which leads htt to become misfolded and kill neuronal cells in Huntington disease (HD). The cytotoxicity of N-terminal mutant htt fragments is evident by severe neurological phenotypes of transgenic mice that express these htt fragments. Clearance of mutant htt is primarily mediated by the ubiquitin-proteasomal sysmtem (UPS) and autophagy. However, the relative efficiency of these two systems to remove toxic forms of mutant htt has not been rigorously compared. Using cellular and mouse models of HD, we found that inhibiting the UPS leads to a greater accumulation of mutant htt than inhibiting autophagy. Moreover, N-terminal mutant htt fragments, but not full-length mutant htt, accumulate in the HD mouse brains after inhibiting the UPS. These findings suggest that the UPS is more efficient than autophagy to remove N-terminal mutant htt.

  1. Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

    Science.gov (United States)

    Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

    2018-02-02

    There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

  2. Saccharomyces cerevisiae aldolase mutants.

    OpenAIRE

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldol...

  3. Design and Pharmacological Characterization of Inhibitors of Amantadine-Resistant Mutants of the M2 Ion Channel of Influenza A Virus¶

    Science.gov (United States)

    Balannik, Victoria; Wang, Jun; Ohigashi, Yuki; Jing, Xianghong; Magavern, Emma; Lamb, Robert A.; DeGrado, William F.; Pinto, Lawrence H.

    2009-01-01

    The A/M2 proton channel of influenza A virus is a target for the anti-influenza drugs amantadine and rimantadine, whose effectiveness was diminished by the appearance of naturally occurring point mutants in the A/M2 channel pore, among which the most common are S31N, V27A and L26F. We have synthesized and characterized the properties of a series of compounds, originally derived from the A/M2 inhibitor BL-1743. A lead compound emerging from these investigations, spiro[5.5]undecan-3-amine, is an effective inhibitor of wild type A/M2 channels and L26F and V27A mutant ion channels in vitro, and also inhibits replication of recombinant mutant viruses bearing these mutations in plaque reduction assays. Differences in the inhibition kinetics between BL-1743, known to bind inside the A/M2 channel pore, and amantadine were exploited to demonstrate competition between these compounds; consistent with the conclusion that amantadine binds inside the channel pore. Inhibition by all of these compounds was shown to be voltage-independent, suggesting that their charged groups within the N-terminal half of the pore, prior to the selectivity filter that defines the region over which the transmembrane potential occurs. These findings not only help define the location and mechanism of binding of M2 channel-blocking drugs, but also demonstrate the feasibility of discovering new inhibitors that target this binding site in a number of amantadine-resistant mutants. PMID:19905033

  4. Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS.

    Science.gov (United States)

    Østergaard, Michael E; Southwell, Amber L; Kordasiewicz, Holly; Watt, Andrew T; Skotte, Niels H; Doty, Crystal N; Vaid, Kuljeet; Villanueva, Erika B; Swayze, Eric E; Bennett, C Frank; Hayden, Michael R; Seth, Punit P

    2013-11-01

    Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.

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

    Science.gov (United States)

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

  6. Morphological mutants of garlic

    Energy Technology Data Exchange (ETDEWEB)

    Choudhary, A.D.; Dnyansagar, V.R. (Nagpur Univ. (India). Dept. of Botany)

    1982-01-01

    Cloves of garlic (Allium sativuum Linn.) were exposed to gamma rays with various doses and different concentrations of ethylmethane sulphonate (EMS), diethyl sulphate (dES) and ethylene imine (EI). In the second and third generations, 16 types of morphological mutants were recorded with varied frequencies. Of all the mutagens used, gamma rays were found to be the most effective in inducing the maximum number of mutations followed EI, EMS and dES in that order.

  7. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    Eric C Beyer

    2013-04-01

    Full Text Available The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8 have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death and formation of cytoplasmic accumulations (that may act as light scattering particles. These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

  8. Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Kjaersgård, I V; Wiborg, O

    1995-01-01

    All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches. Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism. Gly94 and Gly126...... for GDP over GTP. Furthermore the [G94A]EF-Tu mutant possesses an increased GTPase activity. The aminoacyl-tRNA affinity is much reduced for [G94A]EF-Tu, as reflected in an increase of the dissociation rate constant for the ternary complex by a factor of 40. Surprisingly, however, both mutants...... in their GDP forms have a low, but significant affinity for aminoacyl-tRNA, which is not seen for the wild-type elongation factor Tu. The mutants only exhibit minor changes compared to the wild type with respect to in vitro translation of a poly(U) messenger. Udgivelsesdato: 1995-Feb-15...

  9. Aequorin mutants with increased thermostability.

    Science.gov (United States)

    Qu, Xiaoge; Rowe, Laura; Dikici, Emre; Ensor, Mark; Daunert, Sylvia

    2014-09-01

    Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.

  10. Wild Accessions and Mutant Resources

    DEFF Research Database (Denmark)

    Kawaguchi, Masayoshi; Sandal, Niels Nørgaard

    2014-01-01

    Lotus japonicus, Lotus burttii, and Lotus filicaulis are species of Lotus genus that are utilized for molecular genetic analysis such as the construction of a linkage map and QTL analysis. Among them, a number of mutants have been isolated from two wild accessions: L. japonicus Gifu B-129 and Miy...

  11. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  12. Mildew-resistant mutants induced in North American two- and six-rowed malting barley cultivars

    DEFF Research Database (Denmark)

    Molina-Cano, J.L.; Simiand, J.P.; Sopena, A.

    2003-01-01

    and were shown to have two new alleles at the mlo locus; these were designated, respectively, mlo31 and mlo32. Mutant URS2, showing partial resistance, was inherited as a dominant gene, but was not an allele at the Mla locus. The mean yield of each mutant was higher than that of its parental line...

  13. Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, P. Manish [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 (India); Brannigan, James A., E-mail: jab@ysbl.york.ac.uk [York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom); Prabhune, Asmita; Pundle, Archana [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 (India); Turkenburg, Johan P.; Dodson, G. Guy [York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom); Suresh, C. G., E-mail: jab@ysbl.york.ac.uk [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008 (India)

    2005-01-01

    The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants. The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.

  14. Identification of An Arsenic Tolerant Double Mutant With a Thiol-Mediated Component And Increased Arsenic Tolerance in PhyA Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Sung, D.Y.; Lee, D.; Harris, H.; Raab, A.; Feldmann, J.; Meharg, A.; Kumabe, B.; Komives, E.A.; Schroeder, J.I.; /SLAC, SSRL /Sydney U. /Aberdeen U. /UC, San Diego

    2007-04-06

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  15. ADME-guided design and synthesis of aryloxanyl pyrazolone derivatives to block mutant superoxide dismutase 1 (SOD1) cytotoxicity and protein aggregation: potential application for the treatment of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Chen, Tian; Benmohamed, Radhia; Kim, Jinho; Smith, Karen; Amante, Daniel; Morimoto, Richard I; Kirsch, Donald R; Ferrante, Robert J; Silverman, Richard B

    2012-01-12

    Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. The arylsulfanyl pyrazolone (ASP) scaffold was one of the active scaffolds identified in a cell-based high throughput screening assay targeting mutant Cu/Zn superoxide dismutase 1 (SOD1) induced toxicity and aggregation as a marker for ALS. The initial ASP hit compounds were potent and had favorable ADME properties but had poor microsomal and plasma stability. Here, we identify the microsomal metabolite and describe synthesized analogues of these ASP compounds to address the rapid metabolism. Both in vitro potency and pharmacological properties of the ASP scaffold have been dramatically improved via chemical modification to the corresponding sulfone and ether derivatives. One of the ether analogues (13), with superior potency and in vitro pharmacokinetic properties, was tested in vivo for its pharmacokinetic profile, brain penetration, and efficacy in an ALS mouse model. The analogue showed sustained blood and brain levels in vivo and significant activity in the mouse model of ALS, thus validating the new aryloxanyl pyrazolone scaffold as an important novel therapeutic lead for the treatment of this neurodegenerative disorder.

  16. Incomplete flagellar structures in Escherichia coli mutants.

    OpenAIRE

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative ...

  17. Design

    DEFF Research Database (Denmark)

    Volf, Mette

    This publication is unique in its demystification and operationalization of the complex and elusive nature of the design process. The publication portrays the designer’s daily work and the creative process, which the designer is a part of. Apart from displaying the designer’s work methods...... and design parameters, the publication shows examples from renowned Danish design firms. Through these examples the reader gets an insight into the designer’s reality....

  18. Evaluation of high yielding mutants of Hordeumvulgare cultivar Izgrev

    Directory of Open Access Journals (Sweden)

    B. Dyulgerova

    2017-06-01

    Full Text Available Abstract. Seeds of Hordeum vulgare L. cultivar Izgrev were treated with different concentrations of sodium azide to induce genetic variability for the selection of genotypes with improved traits. After passing through different stages of selection, 18 promising mutants were selected for further studies. Eighteen mutants and their parent and national standard cultivar Veslets were evaluated in Complete Block Design with four replications. The research was conducted in 2013 – 2014 and 2014 – 2015 growing seasons in the experimental field of the Institute of Agriculture Karnobat, Southeastern Bulgaria. The characters studied included days to heading, plant height, lodging, peduncle length, spike length, awn length, spikelet number per spike, grain number per spike, grain weight per spike, 1000 grains weight and grain yield. Wide variation among mutant lines was observed for different traits. Mutant lines M4/16 and M 3/14 produced significantly greater grain yield than the parent and standard cultivar. Positive changes in lodging tolerance, grain number per spike, grain weight per spike, 1000 grains weightwere also observed. This study showed positive effects in the use of mutation in inducing improvement for grain yield and some yield related traits.

  19. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    The induced mutagenesis method for deriving pigment mutants of a green microalga, Chlamydomonas reinhardtii CC-124 and their pigment composition as well as ability to assess mutability of contaminated aquatic ecosystems were studied. In the present study, 14086 mutants (colonies) were obtained by exposure of the ...

  20. Cadmium-Sensitive Mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Howden, R; Cobbett, C S

    1992-09-01

    A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (gamma-glutamylcysteine)(n)-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd.

  1. Induced High Lysine Mutants in Barley

    DEFF Research Database (Denmark)

    Doll, Hans; Køie, B.; Eggum, B. O.

    1974-01-01

    Screening of mutagenically treated materials by combined Kjeldahl nitrogen and dye-binding capacity determinations disclosed fourteen barley mutants, which have from a few to about 40 per cent more lysine in the protein and one mutant with 10 per cent less lysine in the protein than the parent...

  2. Los mutantes de la escuela

    Directory of Open Access Journals (Sweden)

    Diego Armando Jaramillo-Ocampo

    2013-01-01

    Full Text Available El presente artículo muestra los resultados parciales del estudio “Juegos en el recreo escolar: un escenario para la formación ciudadana”, cuya pretensión fue comprender los imaginarios sociales de juego en el recreo escolar y su relación con la convivencia social desde la proximidad del enfoque de complementariedad y el diseño de investigación emergente, planteado por Murcia y Jaramillo (2008. Se presentan los desarrollos logrados en dos categorías centrales del estudio: el patio y el cuerpo; dos categorías que mutan constantemente como entidades vivas en la escuela, hacia la configuración de sujetos que reconocen en el otro y lo otro su posibilidad. La escuela viva, donde es posible “ser en relación con”… se reduce a un espacio temporal y físico, limitado por la campana, “el recreo”. El texto muestra, desde la voz de los actores, esa vida que se da y se quita en la escuela y que se posiciona como una más de las imposiciones normalizadas para controlar. Reconoce, finalmente, una propuesta desde la posibilidad que estos dos mutantes propician para una escuela libre y dinámica.

  3. Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

    Science.gov (United States)

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; Winkler, Robert; León-Ramírez, Claudia G; Ruiz-Herrera, José

    2012-03-01

    Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell.

  4. Design

    DEFF Research Database (Denmark)

    Jensen, Ole B.; Pettiway, Keon

    2017-01-01

    by designers, planners, etc. (staging from above) and mobile subjects (staging from below). A research agenda for studying situated practices of mobility and mobilities design is outlined in three directions: foci of studies, methods and approaches, and epistemologies and frames of thinking. Jensen begins......In this chapter, Ole B. Jensen takes a situational approach to mobilities to examine how ordinary life activities are structured by technology and design. Using “staging mobilities” as a theoretical approach, Jensen considers mobilities as overlapping, actions, interactions and decisions...... with a brief description of how movement is studied within social sciences after the “mobilities turn” versus the idea of physical movement in transport geography and engineering. He then explains how “mobilities design” was derived from connections between traffic and architecture. Jensen concludes...

  5. Design

    DEFF Research Database (Denmark)

    Volf, Mette

    Design - proces & metode iBog®  er enestående i sit fokus på afmystificering og operationalisering af designprocessens flygtige og komplekse karakter. Udgivelsen går bag om designerens daglige arbejde og giver et indblik i den kreative skabelsesproces, som designeren er en del af. Udover et bredt...... indblik i designerens arbejdsmetoder og designparametre giver Design - proces & metode en række eksempler fra anerkendte designvirksomheder, der gør det muligt at komme helt tæt på designerens virkelighed....

  6. Butyric acid tolerance of rice mutant M4 families

    Directory of Open Access Journals (Sweden)

    Mauricio Marini Kopp

    2007-01-01

    Full Text Available Hydromorphic soils have a low drainage capacity and are used mainly for the cultivation of irrigated rice.This condition favors the development of anaerobic microorganisms that produce phytotoxic substances. The objective of thisstudy was to evaluate the response of rice mutants to the phytotoxicity caused by butyric acid under anaerobic conditions. Theexperiment consisted of four treatments arranged in a randomized block design. Plants of 40 families were grown in ahydroponic system and the measured variables were root length and length of aerial part (LAP, number of roots (NR androot dry matter (RDM and aerial part dry matter (DMAP. The analysis of variance was performed, the relative performancecalculated and linear regressions were fitted. Only the treatment effect for NR and effect of interaction for LAP were notsignificant. Root length was most affected by the acid and the regressions expressed positive as well as negative effects for acidtolerance in the mutant families.

  7. Design

    Science.gov (United States)

    Buchanan, Richard; Cross, Nigel; Durling, David; Nelson, Harold; Owen, Charles; Valtonen, Anna; Boling, Elizabeth; Gibbons, Andrew; Visscher-Voerman, Irene

    2013-01-01

    Scholars representing the field of design were asked to identify what they considered to be the most exciting and imaginative work currently being done in their field, as well as how that work might change our understanding. The scholars included Richard Buchanan, Nigel Cross, David Durling, Harold Nelson, Charles Owen, and Anna Valtonen. Scholars…

  8. Responses to novelty in staggerer mutant mice.

    Science.gov (United States)

    Misslin, R; Cigrang, M; Guastavino, J M

    1986-01-01

    Responses to novelty in normal C57BL/6 and staggerer mutant mice were recorded. The normal mice confronted a novel object in their familiar environment showed avoidance and burying responses while the staggerer mutant mice contacted it. When given the opportunity to move around freely in simultaneously presented novel and familiar environments, the mutant mice more quickly entered the novel areas than normal animals. these data reveal a significant decrease in the neophobic components of the neotic behaviour in the staggerer mice. However, since the mutant mice did not show a locomotor deficit, the impairment of neophobia seems not to be due to the gait abnormalities of these animals. The results support the view that the cerebellum may contribute to the organization of complex behaviours. Copyright © 1986. Published by Elsevier B.V.

  9. Characterization of MarR Superrepressor Mutants

    OpenAIRE

    Alekshun, Michael N.; Levy, Stuart B.

    1999-01-01

    MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant pr...

  10. Patulin degradation in saccharomyces cerevisiae: Sensitive mutants.

    Science.gov (United States)

    Thonart, P; Sumbu, Z L; Bechet, J

    1985-03-01

    The present experiments (sensitive mutants and transient inhibition of growth) are compatible with the synthesis of an inductible detoxifying substance in the wild type strain. This substance could be glutathione because glutathione detoxification scheme essentially involves properties of the SH group and it is well known that patulin reacts with sulfhy dril groups.Studies are presently being carried out with sensitive mutants to establish definitively the relation between intracellular pool of glutathone and the resistance mechanism of a yeast to patulin.

  11. High-content screening of yeast mutant libraries by shotgun lipidomics

    DEFF Research Database (Denmark)

    Tarasov, Kirill; Stefanko, Adam; Casanovas, Albert

    2014-01-01

    To identify proteins with a functional role in lipid metabolism and homeostasis we designed a high-throughput platform for high-content lipidomic screening of yeast mutant libraries. To this end, we combined culturing and lipid extraction in 96-well format, automated direct infusion...... factor KAR4 precipitated distinct lipid metabolic phenotypes. These results demonstrate that the high-throughput shotgun lipidomics platform is a valid and complementary proxy for high-content screening of yeast mutant libraries....

  12. Copper-sensitive mutant of Arabidopsis thaliana.

    Science.gov (United States)

    van Vliet, C; Anderson, C R; Cobbett, C S

    1995-11-01

    A Cu-sensitive mutant, cup1-1, of Arabidopsis thaliana has a pattern of heavy-metal sensitivity different from that of the cad1 and cad2 mutants, which are deficient in phytochelatin biosynthesis. The latter are significantly sensitive to Cd and Hg and only slightly sensitive to Cu, whereas the cup1-1 mutant is significantly sensitive to Cu, slightly sensitive to Cd, and not more sensitive to Hg, compared to the wild type. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus, which has been mapped to chromosome 1. Genetic and biochemical studies demonstrate that the cup1-1 mutant is not affected in phytochelatin biosynthesis or function. The sensitive phenotype of the cup1-1 mutant is associated with, and probably due to, increased accumulation of higher levels of Cd and Cu compared with the wild type. Consistent with this, a Cu-inducible, root-specific metallothionein gene, MT2a, is expressed in cup1-1 roots under conditions in which it is not expressed in the wild type. Undifferentiated cup1-1 callus tissue did not show the Cu-sensitive phenotype, suggesting that the mutant phenotype, in contrast to cad1 and cad2, is not expressed at the cellular level.

  13. Allele-selective suppression of mutant genes in polyglutamine diseases.

    Science.gov (United States)

    Liu, Chia-Rung; Cheng, Tzu-Hao

    2015-01-01

    Polyglutamine (polyQ) diseases are heritable dominant neurological disorders, caused by abnormal CAG tri-nucleotide expansion in the coding sequence of affected genes. Extension of CAG repeats results in the production of aberrant gene products that are deleterious to neurons, such as transcripts with a CAG stem-loop secondary structure, and proteins containing a long stretch of polyQ residues. Thus, determining methods for the prevention or elimination of these mutant gene products from neuronal cells and translating this knowledge to clinical application are currently important goals in the fields of neurology and neurogenetics. Recently, several studies have revealed intriguing findings related to the allele-selective regulation of CAG-expanded genes, and have proposed novel designs to selectively diminish the mutant polyQ proteins. In this review, we focus on the genes, genetically engineered proteins, and oligonucleotides that show potential to modulate the expression of mutant genes. We also discuss their respective molecular functions at the levels of transcription, translation, and post-translation.

  14. The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control

    NARCIS (Netherlands)

    Wezel, Gilles P. van; Takano, Eriko; Vijgenboom, Erik; Bosch, Leendert; Bibb, Mervyn J.

    1995-01-01

    In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and

  15. Plasmodium berghei: in vivo generation and selection of karyotype mutants and non-gametocyte producer mutants

    NARCIS (Netherlands)

    Janse, C. J.; Ramesar, J.; van den Berg, F. M.; Mons, B.

    1992-01-01

    We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals

  16. Morphological Structure and Genetic Mapping of New Leaf-Color Mutant Gene in Rice (Oryza sativa

    Directory of Open Access Journals (Sweden)

    Yu-hong LI

    2012-06-01

    Full Text Available Leaf-color mutations are a widely-observed class of mutations, playing an important role in the study of chlorophyll biosynthesis and plant chloroplast structure, function, genetics and development. A naturally-occurring leaf-color rice mutant, Baihuaidao 7, was analyzed. Mutant plants typically exhibited a green-white-green leaf-color progression, but this phenotype was only expressed in the presence of a stress signal induced by mechanical scarification such as transplantation. Prior to the appearance of white leaves, mutant plant growth, leaf color, chlorophyll content, and chloroplast ultrastructure appeared to be identical to those of the wild type. After the changeover to white leaf color, an examination of the mutated leaves revealed a decrease in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid content, a reduction in the number of chloroplast grana lamella and grana, and a gradual degradation of the thylakoid lamellas. At maturity, the mutant plant was etiolated and dwarfed compared with wild-type plants. Genetic analysis indicated that the leaf mutant character is controlled by a recessive nuclear gene. Genetic mapping of the mutant gene was performed using an F2 population derived from a Baihuaidao 7 × Jiangxi 1587 cross. The mutant gene was mapped to rice chromosome 11, positioned between InDel markers L59.2-7 and L64.8-11, which are separated by approximately 740.5 kb. The mutant gene is believed to be a new leaf-color mutant gene in rice, and is tentatively designated as gwgl.

  17. Design of thermolabile bacteriophage repressor mutants by comparative molecular modeling

    NARCIS (Netherlands)

    Nauta, A; vandenBurg, B; Karsens, H; Venema, G; Kok, J; Burg, Bertus van den

    1997-01-01

    Comparative molecular modeling was performed with repressor protein Rro of the temperate Lactococcus lactis bacteriophage r1t using the known 3D-structures of related repressors in order to obtain thermolabile derivatives of Rro. Rro residues presumed to stabilize a nonhomologous but structurally

  18. Targeting mutant NRAS signaling pathways in melanoma.

    Science.gov (United States)

    Vu, Ha Linh; Aplin, Andrew E

    2016-05-01

    Cutaneous melanoma is a devastating form of skin cancer and its incidence is increasing faster than any other preventable cancer in the United States. The mutant NRAS subset of melanoma is more aggressive and associated with poorer outcomes compared to non-NRAS mutant melanoma. The aggressive nature and complex molecular signaling conferred by this transformation has evaded clinically effective treatment options. This review examines the major downstream effectors of NRAS relevant in melanoma and the associated advances made in targeted therapies that focus on these effector pathways. We outline the history of MEK inhibition in mutant NRAS melanoma and recent advances with newer MEK inhibitors. Since MEK inhibitors will likely be optimized when combined with other targeted therapies, we focus on recently identified targets that can be used in combination with MEK inhibitors. Published by Elsevier Ltd.

  19. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection.

  20. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  1. Aging Kit mutant mice develop cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Lei Ye

    Full Text Available Both bone marrow (BM and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+ cells counts and ii. the stability of left ventricular (LV contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography in two groups of Kit mutant (W/Wv and W41/W42 and in wild type (WT mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF and LV fractional shortening rates (FS were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

  2. Ovarian abnormalities in the staggerer mutant mouse.

    Science.gov (United States)

    Guastavino, Jean-Marie; Boufares, Salima; Crusio, Wim E

    2005-08-24

    Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rora(sg) gene underlying the cerebellar abnormalities of the staggerer mutant.

  3. Ovarian Abnormalities in the Staggerer Mutant Mouse

    Directory of Open Access Journals (Sweden)

    Jean-Marie Guastavino

    2005-01-01

    Full Text Available Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rorasg gene underlying the cerebellar abnormalities of the staggerer mutant.

  4. Hepatitis B surface gene 145 mutant as a minor population in hepatitis B virus carriers

    Directory of Open Access Journals (Sweden)

    Komatsu Haruki

    2012-01-01

    Full Text Available Abstract Background Hepatitis B virus (HBV can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145 is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted. Methods The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years; group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years. To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products. Results By mutant-specific real-time PCR, one subject (5.6% was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6% of the group 1 subjects and 10 (9.3% of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects of 12 subjects who had overlapped peaks at nt 587 in the electropherogram. Conclusions The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in

  5. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

    Science.gov (United States)

    Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

    2016-01-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  6. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    Science.gov (United States)

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  7. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    The result of bio-test, using the resulting pigment mutant of C. reinhardtii 124y-1 showed that mutagenic activity was observed significantly in both Tekeli River and Pavlodar Oil Refinery in Kazakhstan; the waste water of the. Pavlodar Oil Refinery had high-toxicity while the water of the Tekeli River had medium-toxicity.

  8. Avirulent mutants of Macrophomina phaseolina and Aspergillus ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 25; Issue 1. Avirulent mutants of Macrophomina phaseolina and Aspergillus fumigatus initiate infection in Phaseolus mungo in the presence of phaseo-linone; levamisole gives protection. Suchandra Sett Santosh K Mishra Kazia I Siddiqui. Articles Volume 25 Issue 1 March ...

  9. Ethanol production using engineered mutant E. coli

    Science.gov (United States)

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  10. Flocculation phenomenon of a mutant flocculent Saccharomyces ...

    African Journals Online (AJOL)

    Flocculation phenomenon of a mutant flocculent Saccharomyces cerevisiae strain: Effects of metal ions, sugars, temperature, pH, protein-denaturants and ... was in the early stationary growth phase, which coincided with glucose depletion in the batch fermentation for the production of ethanol from kitchen refuse medium.

  11. Mutant PTEN in Cancer : Worse Than Nothing

    NARCIS (Netherlands)

    Leslie, Nick R; den Hertog, Jeroen

    2014-01-01

    Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant

  12. Fibrinolytic Activity of Recombinant Mutant Streptokinase

    Directory of Open Access Journals (Sweden)

    Mahboobeh Mobarrez

    2015-04-01

    Full Text Available Background: Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using streptokinase is antibody formation which causes allergic reaction to neutralize effects of streptokinase therapy. Aim of this study was investigate of recombinant mutant streptokinase fibrinolytic activity.Materials and Methods: In this study recombinant mutant streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied.Results: The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using zymography, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form.Conclusion: It was found that this product had the more volume and more enzymatic activity.

  13. Characterization of a Legionella micdadei mip mutant

    DEFF Research Database (Denmark)

    O'Connell, W A; Bangsborg, Jette Marie; Cianciotto, N P

    1995-01-01

    The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human m...... Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry...... into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei....

  14. Construction of Comprehensive Dosage-Matching Core Histone Mutant Libraries forSaccharomyces cerevisiae.

    Science.gov (United States)

    Jiang, Shuangying; Liu, Yan; Wang, Ann; Qin, Yiran; Luo, Maoguo; Wu, Qingyu; Boeke, Jef D; Dai, Junbiao

    2017-12-01

    Saccharomyces cerevisiae contains two genes for each core histone, which are presented as pairs under the control of a divergent promoter, i.e. , HHT1-HHF1 , HHT2-HHF2 , HTA1-HTB1 and HTA2-HTB2 HHT1-HHF1 , and HHT2-HHF2 encode histone H3 and H4 with identical amino acid sequences but under the control of differently regulated promoters. Previous mutagenesis studies were carried out by deleting one pair and mutating the other one. Here, we present the design and construction of three additional libraries covering HTA1-HTB1 , HTA2-HTB2 , and HHT1-HHF1 respectively. Together with the previously described library of HHT2-HHF2 mutants, a systematic and complete collection of mutants for each of the eight core S. cerevisiae histone genes becomes available. Each designed mutant was incorporated into the genome, generating three more corresponding libraries of yeast strains. We demonstrated that, although, under normal growth conditions, strains with single-copy integrated histone genes lacked phenotypes, in some growth conditions, growth deficiencies were observed. Specifically, we showed that addition of a second copy of the mutant histone gene could rescue the lethality in some previously known mutants that cannot survive with a single copy. This resource enables systematic studies of function of each nucleosome residue in plasmid, single-copy, and double-copy integrated formats. Copyright © 2017 by the Genetics Society of America.

  15. A new neurological rat mutant "mutilated foot".

    OpenAIRE

    Jacobs, J M; Scaravilli, F; Duchen, L W; Mertin, J

    1981-01-01

    A new autosomal recessive mutant rat (mutilated foot) with a neurological disorder is described. Affected animals become ataxic and the feet, generally of the hind limbs, are mutilated. Quantitative studies show a severe reduction in numbers of sensory ganglion cells and fibres, including unmyelinated fibres. The numbers of ventral root fibres, particularly those of small diameter, are also reduced. Markedly decreased numbers of spindles are found in the limb muscles. These quantitative abnor...

  16. Courtship song analysis of Drosophila muscle mutants.

    Science.gov (United States)

    Chakravorty, Samya; Wajda, Mathew P; Vigoreaux, Jim O

    2012-01-01

    As part of the mating ritual, males of Drosophila species produce species-specific courtship songs through wing vibrations generated by the thoracic musculature. While previous studies have shown that indirect flight muscles (IFM) are neurally activated during courtship song production, the precise role of these muscles in song production has not been investigated. Fortunately, IFM mutants abound in Drosophila melanogaster and studies spanning several decades have shed light on the role of muscle proteins in IFM-powered flight. Analysis of courtship songs in these mutants offers the opportunity to uncover the role of the IFM in a behavior distinct than flight and subject to different evolutionary selection regimes. Here, we describe protocols for the recording and analysis of courtship behavior and mating song of D. melanogaster muscle transgenic and mutant strains. To record faint acoustic signal of courtship songs, an insulated mating compartment was used inside a recording device (INSECTAVOX) equipped with a modified electret microphone, a low-noise power supply, and noise filters. Songs recorded in the INSECTAVOX are digitized using Goldwave, whose several features enable extraction of critical song parameters, including carrier frequencies for pulse song and sine song. We demonstrate the utility of this approach by showing that deletion of the N-terminal region of the myosin regulatory light chain, a mutation known to decrease wing beat frequency and flight power, affects courtship song parameters. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Agrobacterium rhizogenes mutants that fail to bind to plant cells.

    OpenAIRE

    Crews, J L; Colby, S; Matthysse, A G

    1990-01-01

    Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria ...

  18. Isolation and Characterization of Sexual Sporulation Mutants of Aspergillus nidulans

    NARCIS (Netherlands)

    Swart, K.; Heemst, van D.; Slakhorst, M.; Debets, A.J.M.; Heyting, C.

    2001-01-01

    For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with

  19. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants

    NARCIS (Netherlands)

    García-Contreras, R; Lira-Silva, E; Jasso-Chávez, R; Hernández-González, I.L.; Maeda, T.; Hashimoto, T.; Boogerd, F.C.; Sheng, L; Wood, TK; Moreno-Sánchez, R

    2013-01-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed

  20. Strain improvement in dye decolourising mutants of Mucor mucedo ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... The amounts of protoplasts obtained in the developed mutants of M. mucedo MMM1 (U.V. irradiated mutant) and MMM2 (ethyl methyl sulfonate treated mutant) which are very effective decolourisers were. 5.23 x 106 and 5.65 x 106 protoplasts/ml respectively. Among the 385 colonies isolated after ...

  1. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ... some organelles degradation, and membranous multilamellar appear at different stages. Protein and amino acid contents in seeds of mutant are higher than those of the wild type, especially methionine and cysteine. These results suggest that the curled-cotyledons mutant is a novel cotyledon development mutant, which ...

  2. Evaluation of Mungbean Mutant Lines to Drought Stress and Their Genetic Relationships Using SSR Markers

    Directory of Open Access Journals (Sweden)

    Yuliasti

    2015-12-01

    Full Text Available Development of mungbean cultivarstolerant to drought stress through mutation breeding approach would enable us to anticipate the crop yield-reducing effects of climate changes. The objective of this research was to evaluate the yield performance of mungbean mutant lines that showed tolerance to drought stress, and to analyze their genetic diversity and relationship among mutant lines using SSR markers. The study was conducted during the dry season of 2012 in the Muneng experimental farm, Probolinggo, East Java. The experiment was laid out in a randomized block design with four replications. Five mutant lines and two parental lines as control were tested for evaluation of yield and drought tolerance under twoenvironments of two irrigation systems as treatment. The two environmental conditions consisted of optimal irrigation (at least three times: at planting, flowering and during pod filling and suboptimal irrigation (two times at planting and flowering. To evaluate genetic variation among selected mutant lines and their discrimination from parental lines in molecular level, a cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA in the NTSYS software. The results showed that three mutant lines, including PsJ30, PsJ31, PsJ32 produced the highest grain yields of 1.17, 1.01, and 1.04 ton/ha, respectively, compared to the other mutant lines and the parents Gelatik (0.85 ton/ha and Perkutut (0.87 ton/ha as control check. Of those mutant lines, PSJ31 was the most tolerant to drought with sensitivity index value of 0.47. The PSJ31 has now been officially released as a new variety ( 2013, named as Muri which was identified to have high yield and tolerant to drought. Based on 23 SSR markers used for clustering analysis of those 3 selected mutant lines,9SSR markers (MBSS R033; satt137; MBSSR008; MBSSR203; MBSSR013; MBSSR021; MBSSR016; MBSSR136; and DMBSSR013 were successfully identified the three mungbean mutant

  3. Isolation and growth characterization of chlorate and/or bromate resistant mutants generated by spontaneous and induced foreword mutations at several gene loci in aspergillus niger

    Science.gov (United States)

    Kanan, Ghassan J. M.; Al-Najjar, Heyam E.

    2010-01-01

    We aimed her mainly to evaluate the contribution of newly employed bromate selection system, in obtaining new Aspergillus niger nitrate/nitrite assimilation defective mutants, through Ultraviolet treatment (UV), 1, 2, 7, 8-Diepoxyoctane (DEO), phenols mixture (Phx)) and spontaneous treatments. The newly employed bromate selection system was able to specify only two putative novel mutant types designated brn (bromate resistant but chlorate sensitive (RS) strain, which may specify nitrite specific transporter) and cbrn mutants (bromate resistant and chlorate resistant strain, which may specify nitrate/nitrite bispecific system). The most relevant and innovative findings of this research work involve the isolation of the RR ( cbrn) mutants (a new type of nitrate assimilation defective mutants), that could be useful for studying the bispecific nitrate /nitrite transporter system. The majority of obtained bromate resistant mutants (93.3% of the total mutants obtained by all treatments) were of the brn type, whereas the remaining percentage (6.76%) was given to cbrn strains. The highest percentages of brn mutant strains (48% and 58.6% of the total RS strains) were obtained with UA after spontaneous and Phx treatment, whereas Trp has generated 29% and 42% of RS strains after UV and DEO treatments, respectively. The obtained ratios of cbrn mutants were higher (i.e. in the range of 8.4%-11.64% of the total bromate mutants) with chemical treatments, especially when U.A or Pro was serving as sole N-sources at 25ºC rather than 37ºC. A 69% mutants` yield of Aspergillus niger mutant strains representing nine gene loci ( niaD, cnx-6 loci , nrt and nirA) were selected on the bases of chlorate (600 mM) toxicity. All chlorate resistant mutants were completely sensitive to bromate (250 mM). The niaD mutants showed the highest percentage (73.97%) of chlorate resistant mutants obtained with all tested treatments. The UV treatment has generated the highest ratio (86.9%) of nia

  4. Isolation and growth characterization of chlorate and/or bromate resistant mutants generated by spontaneous and induced foreword mutations at several gene loci in Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Ghassan J. M. Kanan

    2010-12-01

    Full Text Available We aimed her mainly to evaluate the contribution of newly employed bromate selection system, in obtaining new Aspergillus niger nitrate/nitrite assimilation defective mutants, through Ultraviolet treatment (UV, 1, 2, 7, 8-Diepoxyoctane (DEO, phenols mixture (Phx and spontaneous treatments. The newly employed bromate selection system was able to specify only two putative novel mutant types designated brn (bromate resistant but chlorate sensitive (RS strain, which may specify nitrite specific transporter and cbrn mutants (bromate resistant and chlorate resistant strain, which may specify nitrate/nitrite bispecific system. The most relevant and innovative findings of this research work involve the isolation of the RR (cbrn mutants (a new type of nitrate assimilation defective mutants, that could be useful for studying the bispecific nitrate /nitrite transporter system. The majority of obtained bromate resistant mutants (93.3% of the total mutants obtained by all treatments were of the brn type, whereas the remaining percentage (6.76% was given to cbrn strains. The highest percentages of brn mutant strains (48% and 58.6% of the total RS strains were obtained with UA after spontaneous and Phx treatment, whereas Trp has generated 29% and 42% of RS strains after UV and DEO treatments, respectively. The obtained ratios of cbrn mutants were higher (i.e. in the range of 8.4%-11.64% of the total bromate mutants with chemical treatments, especially when U.A or Pro was serving as sole N-sources at 25ºC rather than 37ºC. A 69% mutants' yield of Aspergillus niger mutant strains representing nine gene loci (niaD, cnx-6 loci, nrt and nirA were selected on the bases of chlorate (600 mM toxicity. All chlorate resistant mutants were completely sensitive to bromate (250 mM. The niaD mutants showed the highest percentage (73.97% of chlorate resistant mutants obtained with all tested treatments. The UV treatment has generated the highest ratio (86.9% of niaD mutants

  5. Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

    Science.gov (United States)

    2012-01-01

    Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

  6. Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Chen Bo-Ruei

    2012-05-01

    Full Text Available Abstract Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning

  7. TagSmart: analysis and visualization for yeast mutant fitness data measured by tag microarrays

    Directory of Open Access Journals (Sweden)

    Xie Dan

    2007-04-01

    Full Text Available Abstract Background A nearly complete collection of gene-deletion mutants (96% of annotated open reading frames of the yeast Saccharomyces cerevisiae has been systematically constructed. Tag microarrays are widely used to measure the fitness of each mutant in a mutant mixture. The tag array experiments can have a complex experimental design, such as time course measurements and drug treatment with multiple dosages. Results TagSmart is a web application for analysis and visualization of Saccharomyces cerevisiae mutant fitness data measured by tag microarrays. It implements a robust statistical approach to assess the concentration differences among S. cerevisiae mutant strains. It also provides an interactive environment for data analysis and visualization. TagSmart has the following advantages over previously described analysis procedures: 1 it is user-friendly software rather than merely a description of analytical procedure; 2 It can handle complicated experimental designs, such as multiple time points and treatment with multiple dosages; 3 it has higher sensitivity and specificity; 4 It allows users to mask out "bad" tags in the analysis. Two biological tests were performed to illustrate the performance of TagSmart. First, we generated titration mixtures of mutant strains, in which the relative concentration of each strain was controlled. We used tag microarrays to measure the numbers of tag copies in each titration mixture. The data was analyzed with TagSmart and the result showed high precision and recall. Second, TagSmart was applied to a dataset in which heterozygous deletion strain mixture pools were treated with a new drug, Cincreasin. TagSmart identified 53 mutant strains as sensitive to Cincreasin treatment. We individually tested each identified mutant, and found 52 out of the 53 predicted mutants were indeed sensitive to Cincreasin. Conclusion TagSmart is provided "as is" to analyze tag array data produced by Affymetrix and Agilent

  8. Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex

    DEFF Research Database (Denmark)

    Brown, S; Blumenthal, T

    1976-01-01

    of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have...... examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant...... by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly...

  9. HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

    Science.gov (United States)

    On the cover: Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry by the inhibitor. Cover design by Joseph Myer

  10. Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Dolezal, O; Cobbett, C S

    1991-08-01

    A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant.

  11. A ten-week biochemistry lab project studying wild-type and mutant bacterial alkaline phosphatase.

    Science.gov (United States)

    Witherow, D Scott

    2016-11-12

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzymes. The experiments are designed to build on students' work from week to week in a way that requires them to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):555-564, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

  12. Distribution of soluble amino acids in maize endosperm mutants

    Directory of Open Access Journals (Sweden)

    Toro Alejandro Alberto

    2003-01-01

    Full Text Available For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L. mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form.

  13. Using of AFLP to evaluate gamma-irradiated amaranth mutants

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    Labajová Mária

    2013-01-01

    Full Text Available To determine which of several gamma-irradiated mutants of amaranth Ficha cultivar and K-433 hybrid are most genetically similar to their non-irradiated control genotypes, we performed amplified fragment length polymorphism (AFLP based analysis. A total of 40 selective primer combinations were used in reported analyses. First analyses of gamma-irradiated amaranth mutant lines were done used the AFLP. In the study, primers with the differentiation ability for all analysed mutant lines are reported. The very specific changes in the mutant lines´ non-coding regions based on AFLP length polymorphism were analysed. Mutant lines of the Ficha cultivar (C15, C26, C27, C82, C236 shared a genetic dissimilarity of 0,11 and their ISSR profiles are more similar to the Ficha than those of K-433 hybrid mutant lines. The K-433 mutant lines (D54, D279, D282 shared genetic dissimilarity of 0,534 but are more distinct to their control plant as a whole, as those of the Ficha mutant lines. Different AFLP fingerprints patters of the mutant lines when compared to the Ficha cultivar and K-433 hybrid AFLP profiles may be a consequence of the complex response of the intergenic space of mutant lines to the gamma-radiance. Although a genetic polymorphism was detected within accessions, the AFLP markers successfully identified all the accessions. The AFLP results are discussed by a combination of biochemical characteristics of mutant lines and their control genotypes.

  14. Ribosylurea accumulates in yeast urc4 mutants.

    Science.gov (United States)

    Björnberg, O; Vodnala, M; Domkin, V; Hofer, A; Rasmussen, A; Andersen, G; Piskur, J

    2010-06-01

    Yeast Saccharomyces (Lachancea) kluyveri urc4 mutants, unable to grow on uracil, biotransformed (14)C(2)-uracil into two labeled compounds, as detected by high performance liquid chromatography (HPLC). These two compounds could also be obtained following organic synthesis of ribosylurea. This finding demonstrates that in the URC pyrimidine degradation pathway, the opening of the uracil ring takes place when uracil is attached to the ribose moiety. Ribosylurea has not been reported in the cell metabolism before and the two observed compounds likely represent an equilibrium mixture of the pyranosyl and furanosyl forms.

  15. Genetic Analysis and Mapping of TWH Gene in Rice Twisted Hull Mutant

    Directory of Open Access Journals (Sweden)

    Jin-bo LI

    2009-03-01

    Full Text Available A mutant with twisted hulls was found in a breeding population of rice (Oryza sativa L.. The mutant shows less grain weight and inferior grain quality in addition to twisted hulls. Genetic analysis indicated that the phenotype of mutant was controlled by a single recessive gene (temporarily designated as TWH. To map the TWH gene, an F2 population was generated by crossing the twh mutant to R725, an indica rice variety with normal hulls. For bulked segregant analysis, the bulk of mutant plants was prepared by mixing equal amount of plant tissue from 10 twisted-hull plants and the bulk of normal plants was obtained by pooling equal amount tissue of 10 normal-hull plants. Two hundred and seven pairs of simple sequence repeat (SSR primers, which are distributed on 12 rice chromosomes, were used for polymorphism analysis of the parents and the two bulks. The TWH locus was initially mapped close to the SSR marker RM526 on chromosome 2. Therefore, further mapping was performed using 50 pairs of SSR primers around the marker RM526. The TWH was delimited between the SSR markers RM14128 and RM208 on the long arm of chromosome 2 at the genetic distances of 1.4 cM and 2.7 cM, respectively. These results provide the foundation for further fine mapping, cloning and functional analysis of the TWH gene.

  16. Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2010-10-01

    Full Text Available The purpose of the present investigations was to improve the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments viz. UV irradiations, NTG (N-methyl-N-nitro-N-nitrosoguinidine and MMS (methyl methane sulfonate. After screening on skim milk agar plates, a total of nine positive mutants were selected for shake flask experiments. Among these, the best proteolytic mutant designated as UV-9 showed 1.4 fold higher alkaline protease activity in preoptimized growth medium than the parent strain. The fermentation profile and kinetic parameters such u(h-1, Yp/s, Yp/x, Yx/s, q s, Qs, q p and Qp also indicated the superiority of the selected mutant UV-9 for alkaline protease production over the parent strain and rest of the mutants. The dehairing capability of mutant UV-9 alkaline protease was analyzed by soaking goat skin pieces for different time intervals (3-15 h at 40 º C. A complete dehairing without degradation of collagen was achieved after 12 h, indicating its commercial exploitation in leather industry.

  17. Kinase Inhibitor Profiling Reveals Unexpected Opportunities to Inhibit Disease-Associated Mutant Kinases

    Directory of Open Access Journals (Sweden)

    Krisna C. Duong-Ly

    2016-02-01

    Full Text Available Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases, including ALK, LRRK2, RET, and EGFR, as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development.

  18. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.

    Science.gov (United States)

    Rathnaiah, Govardhan; Lamont, Elise A; Harris, N Beth; Fenton, Robert J; Zinniel, Denise K; Liu, Xiaofei; Sotos, Josh; Feng, Zhengyu; Livneh-Kol, Ayala; Shpigel, Nahum Y; Czuprynski, Charles J; Sreevatsan, Srinand; Barletta, Raúl G

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

  19. Molecular characterization of mlo mutants in North American two- and six-rowed malting barley cultivars.

    Science.gov (United States)

    Panstruga, Ralph; Molina-Cano, José Luis; Reinstädler, Anja; Müller, Judith

    2005-05-01

    SUMMARY Barley lines PRU1, URS1 and URS2 represent three candidate mlo mutants induced in either the two-rowed cultivar Prudentia or the six-rowed cultivar Ursula. Both Prudentia and Ursula are North American malting barley varieties with specific malting properties. Here, we analysed the three candidate mutants at the molecular level. We identified lesions in the Mlo gene of all three lines, causing either a premature stop codon (PRU1), a shift in the reading frame (URS1) or a single amino acid replacement (URS2). In a transient gene expression assay, the URS2 mlo allele fails to complement a barley null mutant genotype, indicating that URS2 is a genuine mlo mutant (here designated as mlo-33). The MLO-33 mutant variant accumulates to similar levels as the wild-type MLO protein in Arabidopsis protoplasts, suggesting that MLO-33 is stable in planta. We show that the mlo-33 allele can be readily detected in barley genomic DNA by a cleaved amplified polymorphic sequence marker, rendering this allele particularly suited for marker-assisted breeding.

  20. Developmental defects in mutants of the PsbP domain protein 5 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Johnna L Roose

    Full Text Available Plants contain an extensive family of PsbP-related proteins termed PsbP-like (PPL and PsbP domain (PPD proteins, which are localized to the thylakoid lumen. The founding member of this family, PsbP, is an established component of the Photosystem II (PS II enzyme, and the PPL proteins have also been functionally linked to other photosynthetic processes. However, the functions of the remaining seven PPD proteins are unknown. To elucidate the function of the PPD5 protein (At5g11450 in Arabidopsis, we have characterized a mutant T-DNA insertion line (SALK_061118 as well as several RNAi lines designed to suppress the expression of this gene. The functions of the photosynthetic electron transfer reactions are largely unaltered in the ppd5 mutants, except for a modest though significant decrease in NADPH dehydrogenase (NDH activity. Interestingly, these mutants show striking plant developmental and morphological defects. Relative to the wild-type Col-0 plants, the ppd5 mutants exhibit both increased lateral root branching and defects associated with axillary bud formation. These defects include the formation of additional rosettes originating from axils at the base of the plant as well as aerial rosettes formed at the axils of the first few nodes of the shoot. The root-branching phenotype is chemically complemented by treatment with the synthetic strigolactone, GR24. We propose that the developmental defects observed in the ppd5 mutants are related to a deficiency in strigolactone biosynthesis.

  1. Auxin physiology of the tomato mutant diageotropica

    Science.gov (United States)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  2. Indy mutants: live long and prosper

    Directory of Open Access Journals (Sweden)

    Stewart eFrankel

    2012-02-01

    Full Text Available Indy encodes the fly homologue of a mammalian transporter of di and tricarboxylatecomponents of the Krebs cycle. Reduced expression of fly Indy or two of the C. elegansIndy homologs leads to an increase in life span. Fly and worm tissues that play key roles inintermediary metabolism are also the places where Indy genes are expressed. One of themouse homologs of Indy (mIndy is mainly expressed in the liver. It has been hypothesizedthat decreased INDY activity creates a state similar to caloric restriction (CR. Thishypothesis is supported by the physiological similarities between Indy mutant flies on highcalorie food and control flies on CR, such as increased physical activity and decreases inweight, egg production, triglyceride levels, starvation resistance, and insulin signaling. Inaddition, Indy mutant flies undergo changes in mitochondrial biogenesis also observed inCR animals. Recent findings with mIndy knockout mice support and extend the findingsfrom flies. mIndy-/- mice display an increase in hepatic mitochondrial biogenesis, lipidoxidation and decreased hepatic lipogenesis. When mIndy-/- mice are fed high calorie foodthey are protected from adiposity and insulin resistance. These findings point to INDY as apotential drug target for the treatment of metabolic syndrome, type 2 diabetes and obesity.

  3. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A.

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  4. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  5. Forward genetic screen for auxin-deficient mutants by cytokinin.

    Science.gov (United States)

    Wu, Lei; Luo, Pan; Di, Dong-Wei; Wang, Li; Wang, Ming; Lu, Cheng-Kai; Wei, Shao-Dong; Zhang, Li; Zhang, Tian-Zi; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, Guang-Qin

    2015-07-06

    Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

  6. Design and Discovery of N -(2-Methyl-5'-morpholino-6'-((tetrahydro-2 H -pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers

    Energy Technology Data Exchange (ETDEWEB)

    Nishiguchi, Gisele A.; Rico, Alice; Tanner, Huw; Aversa, Robert J.; Taft, Benjamin R.; Subramanian, Sharadha; Setti, Lina; Burger, Matthew T.; Wan, Lifeng; Tamez, Victoriano; Smith, Aaron; Lou, Yan; Barsanti, Paul A.; Appleton, Brent A.; Mamo, Mulugeta; Tandeske, Laura; Dix, Ina; Tellew, John E.; Huang, Shenlin; Mathews Griner, Lesley A.; Cooke, Vesselina G.; Van Abbema, Anne; Merritt, Hanne; Ma, Sylvia; Gampa, Kalyani; Feng, Fei; Yuan, Jing; Wang, Yingyun; Haling, Jacob R.; Vaziri, Sepideh; Hekmat-Nejad, Mohammad; Jansen, Johanna M.; Polyakov, Valery; Zang, Richard; Sethuraman, Vijay; Amiri, Payman; Singh, Mallika; Lees, Emma; Shao, Wenlin; Stuart, Darrin D.; Dillon, Michael P.; Ramurthy, Savithri

    2017-06-06

    RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [Aversa, Biaryl amide compounds as kinase inhibitors and their preparation. WO 2014151616, 2014], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

  7. Google: a narrativa de uma marca mutante

    Directory of Open Access Journals (Sweden)

    Elizete de Azevedo Kreutz

    2010-01-01

    Full Text Available As marcas mutantes já fazem parte de nossa realidade, embora ainda não totalmente percebidas e/ou aceitas como tal. O presente artigo busca refletir sobre a relevância dessas novas estratégias de comunicação e branding, identificando suas principais características. Para isso, utilizamos o método de estudo de caso, o Google, ancorado nos métodos de pesquisa bibliográfica e de internet. A escolha foi intencional, posto que a organização é referência em sua categoria, mecanismo de busca, e reflete essa estratégia comunicacional contemporânea. Como resultado, as informações obtidas nos possibilitam compreender essa tendência de comportamento de marca que busca a interação com seus públicos.

  8. Studies on mutant breeding of Hibiscus syriacus

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hi Sup; Kim, Jin Kyu; Lee, Ki Un; Kim, Young Taik

    1997-01-01

    Hibiscus has been known as a national flower of Korea. Hibiscus has such a characteristic of self-incompatibility that all the plant exist as natural hybrids and have heterogeneous genes. Many domestic 91 varieties of Hibiscus syriacus were collected. Radiosensitivity of H. Syriacus irradiated with {gamma}-ray was investigated in plant cuttings. The plant height was reduced by 45% in 5KR irradiated group, compared to control group. The radiation dose of 5KR could be recommended for mutation breeding of Hibiscus cuttings. Radiosensitivity of {gamma}-ray irradiated Hibiscus seed were investigated. The germination rate, survival rate and plant height was better in the 4KR irradiation plot than control. The radiation dose of 10{approx}12KR are recommended for mutation breeding of Hibiscus. Promising mutant lines were selected form the varieties of Hwarang, Wolsan no. 176, Ilpyondansim, Emille, Hanol, Yongkwang, Saeyongkwang, Chungmu, Imjinhong, Arang, Hungdansim-1 and Hongdansim-2. (author). 66 refs., 16 tabs., 13 figs.

  9. Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants.

    Science.gov (United States)

    Kumar, Vivek; Kim, Kyungin; Joseph, Chryshanthi; Thomas, Lisa C; Hong, Heekyung; Takahashi, Joseph S

    2011-09-13

    Forward genetic screens have been highly successful in revealing roles of genes and pathways in complex biological events. Traditionally these screens have focused on isolating mutants with the greatest phenotypic deviance, with the hopes of discovering genes that are central to the biological event being investigated. Behavioral screens in mice typically use simple activity-based assays as endophenotypes for more complex emotional states of the animal. They generally set the selection threshold for a putative mutant at 3 SDs (z score of 3) from the average behavior of normal animals to minimize false-positive results. Behavioral screens using a high threshold for detection have generally had limited success, with high false-positive rates and subtle phenotypic differences that have made mapping and cloning difficult. In addition, targeted reverse genetic approaches have shown that when genes central to behaviors such as open field behavior, psychostimulant response, and learning and memory tasks are mutated, they produce subtle phenotypes that differ from wild-type animals by 1 to 2 SDs (z scores of 1 to 2). We have conducted a second-generation (G2) dominant N-ethyl-N-nitrosourea (ENU) screen especially designed to detect subtle behavioral mutants for open field activity and psychostimulant response behaviors. We successfully detect mutant lines with only 1 to 2 SD shifts in mean response compared with wild-type control animals and present a robust statistical and methodological framework for conducting such forward genetic screens. Using this methodology we have screened 229 ENU mutant lines and have identified 15 heritable mutant lines. We conclude that for screens in mice that use activity-based endophenotypic measurements for complex behavioral states, this G2 screening approach yields better results.

  10. Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

    Science.gov (United States)

    2016-11-01

    AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina...5a. CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER...epigenomic landscape of EGFR mutant SCLCs and their corresponding pre- treatment LUADs. These are very rare specimens. Through our Yale rebiopsy program

  11. Mutant-specific gene programs in the zebrafish

    OpenAIRE

    Weber, Gerhard J.; Choe, Sung E; Dooley, Kimberly A.; Paffett-Lugassy, Noëlle N.; Zhou, Yi; Zon, Leonard I.

    2005-01-01

    Hematopoiesis involves the production of stem cells, followed by the orchestrated differentiation of the blood lineages. Genetic screens in zebrafish have identified mutants with defects that disrupt specific stages of hematopoiesis and vasculogenesis, including the cloche, spadetail (tbx16), moonshine (tif1g), bloodless, and vlad tepes (gata1) mutants. To better characterize the blood program, gene expression profiling was carried out in these mutants and in scl-morphants (scl mo). Distinct ...

  12. Mutant p53 in Cancer: New Functions and Therapeutic Opportunities

    Science.gov (United States)

    Muller, Patricia A.J.; Vousden, Karen H.

    2014-01-01

    Many different types of cancer show a high incidence of TP53 mutations, leading to the expression of mutant p53 proteins. There is growing evidence that these mutant p53s have both lost wild-type p53 tumor suppressor activity and gained functions that help to contribute to malignant progression. Understanding the functions of mutant p53 will help in the development of new therapeutic approaches that may be useful in a broad range of cancer types. PMID:24651012

  13. Growth and development of maize that contains mutant tubulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  14. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    Energy Technology Data Exchange (ETDEWEB)

    Brumleve, B.; Lev, M.

    1986-05-01

    Bacteroides levii, an anaerobic bacterium, synthesizes two sphingolipids; the sphingomyelin analogue, ceramide phosphorylethanolamine (CPE), and also ceramide phosphorylglycerol (CPG). The first enzyme in the sphingolipid pathway, 3-ketodihydro-sphingosine (3KDS) synthase, has been partially purified previously. To study subsequent steps in the pathways, mutants defective in sphingolipid synthesis were derived by ethyl methanesulfonate and nitrosoguanidine mutagenesis. Extracts of the mutant, 1075BB, show synthase activity although the cells do not synthesize CPE or CPG. The mutant differs from the wild type in that: (1) synthase activity was much diminished in the mutant, (2) sphingolipid synthesis does not occur in the mutant as evidenced by the absence of spots at sites where CPE and CPG migrate following two-dimensional thin layer chromatography, (3) incorporation of uniformly-labelled (/sup 14/C)serine carbon or (/sup 14/C)3KDS into sphingolipids was not observed in the mutant, (4) following incubation with (/sup 14/C)3KDS, radioactivity corresponding to dihydrosphingosine (DHS) and ceramide were observed in the mutant; no (/sup 14/C)DHS was detected in the wild type, and (5) enhanced incorporation of (/sup 14/C)serine carbon into two lipids not containing phosphorus was found in the mutant. The authors conclude, therefore, that this mutant, 1075BB, has a metabolic block at the terminal biosynthetic steps of sphingolipid synthesis.

  15. Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others.

    Science.gov (United States)

    Sabapathy, Kanaga; Lane, David P

    2017-09-26

    TP53, which encodes the tumour-suppressor protein p53, is the most frequently mutated gene across all cancer types. The presence of mutant p53 predisposes to cancer development, promotes the survival of cancer cells, and is associated with ineffective therapeutic responses and unfavourable prognoses. Despite these effects, no drug that abrogates the oncogenic functions of mutant p53 has yet been approved for the treatment of cancer. Current investigational therapeutic strategies are mostly aimed at restoring the wild-type activity of mutant p53, based on the assumption that all p53 mutants are functionally equal. Our increasing knowledge of mutant forms of p53, however, supports the antithetical hypothesis that not all p53 mutants have equivalent cellular effects; hence, a judicious approach to therapeutic targeting of mutant p53 is required. In this Review, we propose a categorization of the major classes of p53 mutants based on their functionality in tumour suppression and response to therapy. The emerging picture is that the mutations across TP53 form a 'rainbow of mutants', with varying degrees of functionality and different pathobiological consequences, necessitating the use of diverse therapeutic strategies to selectively target specific classes of mutation. The utility of this knowledge of TP53 mutations in developing selective therapeutic options, and in facilitating clinical decision-making is discussed.

  16. Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae.

    Science.gov (United States)

    Hedman, Jamie M; Eggleston, Matthew D; Attryde, Amanda L; Marshall, Pamela A

    2007-10-01

    Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.

  17. Isolation of Bacteriophage T4 Mutants Defective in the Ability to Degrade Host Deoxyribonucleic Acid 1

    Science.gov (United States)

    Warner, Huber R.; Snustad, D. Peter; Jorgensen, Sally E.; Koerner, James F.

    1970-01-01

    A method was devised for identifying nonlethal mutants of T4 bacteriophage which lack the capacity to induce degradation of the deoxyribonucleic acid (DNA) of their host, Escherichia coli. If a culture is infected in a medium containing hydroxyurea (HU), a compound that blocks de novo deoxyribonucleotide biosynthesis by interacting with ribonucleotide reductase, mutant phage that cannot establish the alternate pathway of deoxyribonucleotide production from bacterial DNA will fail to produce progeny. The progeny of 100 phages that survived heavy mutagenesis with hydroxylamine were tested for their ability to multiply in the presence of HU. Four of the cultures lacked this capacity. Cells infected with one of these mutants, designated T4nd28, accumulated double-stranded fragments of host DNA with a molecular weight of approximately 2 × 108 daltons. This mutant failed to induce T4 endonuclease II, an enzyme known to produce single-strand breaks in double-stranded cytosine-containing DNA. The properties of nd28 give strong support to an earlier suggestion that T4 endonuclease II participates in host DNA degradation. The nd28 mutation mapped between T4 genes 32 and 63 and was very close to the latter gene. It is, thus, in the region of the T4 map that is occupied by genes for a number of other enzymes, including deoxycytidylate deaminase, thymidylate synthetase, dihydrofolate reductase, and ribonucleotide reductase, that are nonessential to phage production in rich media. PMID:4914096

  18. Stability Test For Sorghum Mutant Lines Derived From Induced Mutations with Gamma-Ray Irradiation

    Directory of Open Access Journals (Sweden)

    S. Human

    2011-12-01

    Full Text Available Sorghum breeding program had been conducted at the Center for the Application of Isotopes and Radiation Technology, BATAN. Plant genetic variability was increased through induced mutations using gamma-ray irradiation. Through selection process in successive generations, some promising mutant lines had been identified to have good agronomic characteristics with high grain yield. These breeding lines were tested in multi location trials and information of the genotypic stability was obtained to meet the requirements for officially varietal release by the Ministry of Agriculture. A total of 11 sorghum lines and varieties consisting of 8 mutant lines derived from induced mutations (B-100, B-95, B-92, B-83, B-76, B-75, B-69 and Zh-30 and 3 control varieties (Durra, UPCA-S1 and Mandau were included in the experiment. All materials were grown in 10 agro-ecologically different locations namely Gunungkidul, Bantul, Citayam, Garut, Lampung, Bogor, Anyer, Karawaci, Cianjur and Subang. In each location, the local adaptability test was conducted by randomized block design with 3 replications. Data of grain yield was used for evaluating genotypic stability using AMMI approach. Results revealed that sorghum mutation breeding had generated 3 mutant lines (B-100, B-76 and Zh-30 exhibiting grain yield significantly higher than the control varieties. These mutant lines were genetically stable in all locations so that they would be recommended for official release as new sorghum varieties to the Ministry of Agriculture

  19. Isolation and characterization of stable mutants of Streptomyces ...

    Indian Academy of Sciences (India)

    Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer ...

  20. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    These pathways were related to amino acid metabolism, sugar metabolism, and series of major physiological metabolism. Our results hopefully shed light on the further study of molecular mechanism of white egg 2 mutant. Key words: Bombyx mori, white egg 2 mutant, microarray, embryo, differentially expressed gene.

  1. Genomic diversity among Basmati rice ( Oryza sativa L) mutants ...

    African Journals Online (AJOL)

    Genomic diversity among Basmati rice ( Oryza sativa L) mutants obtained through 60 Co gamma radiations using AFLP markers. ... In order to obtain new varieties of rice with improved agronomic and grain characteristics, gamma radiation (60Co) has been used to generate novel mutants of the Basmati rice. In this study ...

  2. Enhanced production of glucose oxidase from UV- mutant of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-01-19

    Jan 19, 2009 ... UV rays were used as mutagen in wild type strain of Aspergillus niger for enhanced production of glucose oxidase. After mutangenization and selection, mutant A. niger strains, resistant to 2-deoxy-D- glucose were obtained. The mutants showed 1.57 and 1.98 fold increase in activities of extra and intra.

  3. Comparison of lignin deposition in three ectopic lignification mutants.

    Science.gov (United States)

    Rogers, Louisa A; Dubos, Christian; Surman, Christine; Willment, Janet; Cullis, Ian F; Mansfield, Shawn D; Campbell, Malcolm M

    2005-10-01

    The Arabidopsis thaliana mutants de-etiolated3 (det3), pom-pom1 (pom1) and ectopic lignification1 (eli1) all deposit lignins in cells where these polymers would not normally be found. Comparison of these mutants provides an opportunity to determine if the shared mutant phenotype arose by perturbing a common regulatory mechanism in each of the mutants. The mutants were compared using a combination of genetics, histochemistry, chemical profiling, transcript profiling using both Northern blots and microarrays, and bioinformatics. The subset of cells that ectopically lignified was shared between all three mutants, but clear differences in cell wall chemistry were evident between the mutants. Northern blot analysis of lignin biosynthetic genes over diurnal and circadian cycles revealed that transcript abundance of several key genes was clearly altered in all three mutants. Microarray analysis suggests that changes in the expression of specific members of the R2R3-MYB and Dof transcription factor families may contribute to the ectopic lignification phenotypes. This comparative analysis provides a suite of hypotheses that can be tested to examine the control of lignin biosynthesis.

  4. Molecularly targeted therapies for p53-mutant cancers.

    Science.gov (United States)

    Zhao, Dekuang; Tahaney, William M; Mazumdar, Abhijit; Savage, Michelle I; Brown, Powel H

    2017-11-01

    The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells.

  5. Unfolding intermediates of the mutant His-107-Tyr of human ...

    Indian Academy of Sciences (India)

    The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulationsand analyzed in ...

  6. Assessment of Genetic diversity in mutant cowpea lines using ...

    African Journals Online (AJOL)

    FKOLADE

    2016-11-09

    Nov 9, 2016 ... for crop improvement, hence the need to broaden the genetic base of any crop. This study was done in order to further enhance this in cowpea. While assessing diversity and phylogenetic relationship with other mutants and their parents, each unique mutant was also characterized. Randomly amplified ...

  7. Cadmium-Sensitive Mutants of Arabidopsis thaliana1

    Science.gov (United States)

    Howden, Ross; Cobbett, Christopher S.

    1992-01-01

    A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (γ-glutamylcysteine)n-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd. Images Figure 1 Figure 7 PMID:16652930

  8. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... egg 2 (w-2) has the same phenotypes as white egg 1 and white egg 3 mutants with white egg color, but its mechanism is more complicated than white egg 1 and white egg 3 mutants based on recent report (Tatematsu et al., 2011) which suggest that the silkworm w-2 locus existed multi-allelic mutations.

  9. Characterization of human glucocerebrosidase from different mutant alleles

    NARCIS (Netherlands)

    Ohashi, T.; Hong, C. M.; Weiler, S.; Tomich, J. M.; Aerts, J. M.; Tager, J. M.; Barranger, J. A.

    1991-01-01

    Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from

  10. Photosynthetic characterization of a rolled leaf mutant of rice ( Oryza ...

    African Journals Online (AJOL)

    A new rolling leaf rice mutant was identified which showed an apparently straighter longitudinal shape normal transverse rolling characters at all developing stages. The chlorophyll contents per fresh weight of this mutant leaves were lower than those of wild-type. The electron transfer rate (ETR) and photochemical ...

  11. Unfolding intermediates of the mutant His-107-Tyr of human ...

    Indian Academy of Sciences (India)

    Srabani Taraphder

    Abstract. The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulations.

  12. Decreased cariogenicity of a mutant of Streptococcus mutans

    NARCIS (Netherlands)

    Stoppelaar, J.D.; König, K.G.; Plasschaert, A.J.M.; Hoeven, J.S. van der

    A strain of Streptococcus mutans was treated with a mutagenic agent. This resulted in isolation of a mutant which, compared to the original strain, had lost the ability to form sticky deposits on hard surfaces in sucrose medium. Apart from colonial morphology, the mutant had not changed in any other

  13. Phospholamban Mutants Compete with Wild Type for SERCA Binding in Living Cells

    OpenAIRE

    Gruber, Simon J.; Haydon, Suzanne; Thomas, David D.

    2012-01-01

    We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca2+ cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigat...

  14. The genetics of white-eye, a sex-linked mutant of Aedes (Stegomyia) cooki Belkin.

    Science.gov (United States)

    Wade, J O

    1977-12-01

    A mutant designated white-eye (w) is described from a colony of Aedes cooki. White-eye is sex-linked and recessive and the crossover value with the sex locus is 7.18 +/- 1.38%. The gene w has full penetrance and expressivity at all stages of the life-cycle, but in the adults the eyes darken with age to a yellowish colour.

  15. Mutants of Cre recombinase with improved accuracy

    Science.gov (United States)

    Eroshenko, Nikolai; Church, George M.

    2013-01-01

    Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wildtype and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M, and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells, and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins. PMID:24056590

  16. Induction and characterization of Arabidopsis mutants by Ion beam

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S. [Gyeongbuk Institute for Bio Industry, Andong (Korea, Republic of)

    2008-03-15

    This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and {gamma}-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

  17. Misfolded opsin mutants display elevated β-sheet structure.

    Science.gov (United States)

    Miller, Lisa M; Gragg, Megan; Kim, Tae Gyun; Park, Paul S-H

    2015-10-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. Copyright © 2015 Federation of European Biochemical Societies. All rights reserved.

  18. Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana 1

    Science.gov (United States)

    Dolezal, Olan; Cobbett, Christopher S.

    1991-01-01

    A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant. ImagesFigure 2Figure 3 PMID:16668327

  19. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    Science.gov (United States)

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Human liver cell trafficking mutants: characterization and whole exome sequencing.

    Directory of Open Access Journals (Sweden)

    Fei Yuan

    Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

  1. PedonnanceofE3rly MatUring MutantS Derived from ''SuPa'~ Rice ...

    African Journals Online (AJOL)

    sig"ificant di.ffere-"~s between the mutants and their'parent for 'ail the maraders testeiiexcept 1 (j()(J grains weight and ~ipe weight The mutants .... reported in earlier rice improvement programmes

  2. Preferência de Bemisia tabaci biótipo B em linhagens mutantes de algodoeiro Bemisia tabaci biotype B preference in mutant cotton lines

    Directory of Open Access Journals (Sweden)

    Francisco das Chagas Vidal Neto

    2008-02-01

    Full Text Available Os efeitos de caracteres mutantes morfológicos do algodoeiro (Gossypium hirsutum L. r. latifolium Hutch.: folha okra, bráctea frego e planta vermelha, em relação à resistência à mosca-branca (Bemisia tabaci biótipo B Hemiptera: Aleyrodidae, foram avaliados em experimentos com ou sem chance de escolha. Os experimentos foram conduzidos em casa-de-vegetação, no delineamento de blocos ao acaso, em fatorial 23 + 1, com quatro repetições. O mutante com a característica planta vermelha foi menos atrativo e menos preferido para oviposição, em relação à planta verde, em ambos os ensaios, com ou sem escolha. Não houve preferência quanto à forma da folha e ao tipo de bráctea.The effects of cotton lines (Gossypium hirsutum L. r. latifolium Hutch. with mutants morphologic characteristics: okra leaf, frego bract and red plant in relation to host plant resistance to whitefly (Bemisia tabaci bioyipe B Hemiptera: Aleyrodidae, were evaluated in choice or no choice assays. The assays were carried out in the greenhouse conditions, according to a completely randomized block design, in a 23 + 1 in a factorial arrangement with four replications. The mutant with red plant characteristic was less attractive and less preferred for oviposition than the normal green plant does, in both, whit or without choice tests. It did not have preference in relation to the form of the leaf and bract type.

  3. Mapping pathological phenotypes in Reelin mutant mice

    Directory of Open Access Journals (Sweden)

    Caterina eMichetti

    2014-09-01

    Full Text Available Autism Spectrum Disorders (ASD are neurodevelopmental disorders with multifactorial origin characterized by social communication and behavioural perseveration deficits. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we investigated the behavioural, neurochemical and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reeler pups. We now report that adult male heterozygous reeler mice did not show social behaviour and communication deficits during male-female social interactions. Wildtype and heterozygous mice also showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection only heterozygous mice showed an over response to stress. At the end of the behavioural studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in heterozygous mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD

  4. Differences in growth promotion, drug response and intracellular protein trafficking of FLT3 mutants.

    Science.gov (United States)

    Mashkani, Baratali; Griffith, Renate; Ashman, Leonie

    2014-11-01

    Mutant forms FMS-like tyrosine kinase-3 (FLT3), are reported in 25% of childhood acute lymphoid leukemia (ALL) and 30% of acute myeloid leukemia (AML) patients. In this study, drug response, growth promoting, and protein trafficking of FLT3 wild-type was compared with two active mutants (Internal Tandem Duplication (ITD)) and D835Y. FLT3 was expressed on factor-dependent cells (FDC-P1) using retroviral transduction. The inhibitory effects of CEP701, imatinib, dasatinib, PKC412 and sunitinib were studied on cell proliferation and FLT3 tyrosine phosphorylation. Total expression and proportion of intracellular and surface FLT3 was also determined. FDC-P1 cells became factor-independent after expression of human FLT3 mutants (ITD and D835Y). FDC-P1 cells expressing FLT3-ITD grow 3 to 4 times faster than those expressing FLT3-D835Y. FD-FLT3-ITD cells were three times more resistant to sunitinib than the FD-FLT3-WT cells. The Geo means for surface FLT3 expression in FD-FLT3-ITD and -D835Y were 65 and 70% less than the FD-FLT3-WT cells. About 40% of expressed FLT3 was detected as intracellular in FD-FLT3-D835Y cell compared to 4 and 4.5% in FD-FLT3-WT and -ITD cells. Retention of D835Y FLT3 mutant protein may cause altered signaling, endoplasmic reticulum stress and activation of apoptotic signaling pathways leading to lower proliferation rate in FD-FLT3-D835Y than the FLT3-WT and ITD mutant., these may also also contribute, along with the preferential affinity, to the increased sensitivity of D835Y of CEP701 and PKC412. Studying these genetic variations can help determining the prognosis and designing a therapeutic plan for the patients with FLT3 mutations.

  5. Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

    Science.gov (United States)

    Perales, Celia; Agudo, Rubén; Domingo, Esteban

    2009-01-01

    Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. (i) When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii) A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii) Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a high dose of the same mutagen, followed by a combination of another mutagen with

  6. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

    Directory of Open Access Journals (Sweden)

    Govardhan eRathnaiah

    2014-10-01

    Full Text Available Mycobacterium avium subsp. paratuberculosis (MAP is the etiologic agent of Johne’s Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95% was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

  7. Phospholamban mutants compete with wild type for SERCA binding in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Gruber, Simon J.; Haydon, Suzanne [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Thomas, David D., E-mail: ddt@umn.edu [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.

  8. Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

    Directory of Open Access Journals (Sweden)

    Celia Perales

    Full Text Available BACKGROUND: Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. METHODOLOGY AND PRINCIPAL FINDINGS: Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. CONCLUSIONS: (i When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a

  9. Modelling the spread of HIV immune escape mutants in a vaccinated population.

    Science.gov (United States)

    Fryer, Helen R; McLean, Angela R

    2011-12-01

    Because cytotoxic T-lymphocytes (CTLs) have been shown to play a role in controlling human immunodeficiency virus (HIV) infection and because CTL-based simian immunodeficiency virus (SIV) vaccines have proved effective in non-human primates, one goal of HIV vaccine design is to elicit effective CTL responses in humans. Such a vaccine could improve viral control in patients who later become infected, thereby reducing onwards transmission and enhancing life expectancy in the absence of treatment. The ability of HIV to evolve mutations that evade CTLs and the ability of these 'escape mutants' to spread amongst the population poses a challenge to the development of an effective and robust vaccine. We present a mathematical model of within-host evolution and between-host transmission of CTL escape mutants amongst a population receiving a vaccine that elicits CTL responses to multiple epitopes. Within-host evolution at each epitope is represented by the outgrowth of escape mutants in hosts who restrict the epitope and their reversion in hosts who do not restrict the epitope. We use this model to investigate how the evolution and spread of escape mutants could affect the impact of a vaccine. We show that in the absence of escape, such a vaccine could markedly reduce the prevalence of both infection and disease in the population. However the impact of such a vaccine could be significantly abated by CTL escape mutants, especially if their selection in hosts who restrict the epitope is rapid and their reversion in hosts who do not restrict the epitope is slow. We also use the model to address whether a vaccine should span a broad or narrow range of CTL epitopes and target epitopes restricted by rare or common HLA types. We discuss the implications and limitations of our findings. © 2011 Fryer, McLean.

  10. Characterization of a Virescent Chloroplast Mutant of Tobacco 1

    Science.gov (United States)

    Archer, E. Kathleen; Bonnett, Howard T.

    1987-01-01

    Virescent mutations produce plants in which young leaves have reduced chlorophyll levels but accumulate nearly normal amounts of chlorophyll as they age; they are predominantly nuclear mutations. We describe here a virescent mutation (designated Vir-c) found in a somatic hybrid line derived from Nicotiana tabacum L. and Nicotiana suaveolens Lehm. This mutation is inherited maternally. Young, half-expanded Vir-c leaves contained three to six times less chlorophyll than did control leaves, and reached maximum chlorophyll levels much later in development. Chlorophyll synthesis rates and chloroplast numbers per cell in Vir-c were similar to the control, and carotenoid content in Vir-c was sufficient to protect chlorophyll from photo-oxidation. Photosynthetic rates of Vir-c at low light intensities suggested a reduced ability to collect light. Electron micrographs of Vir-c chloroplasts from half-expanded leaves showed a significant reduction in thylakoids per granum. The decrease in granal thylakoids was strongly associated with low chlorophyll levels; mature Vir-c leaves with nearly normal chlorophyll content showed normal granal profiles. These results are discussed in relation to virescent mutants previously described. Images Fig. 3 PMID:16665364

  11. Characterization of a virescent chloroplast mutant of tobacco.

    Science.gov (United States)

    Archer, E K; Bonnett, H T

    1987-04-01

    Virescent mutations produce plants in which young leaves have reduced chlorophyll levels but accumulate nearly normal amounts of chlorophyll as they age; they are predominantly nuclear mutations. We describe here a virescent mutation (designated Vir-c) found in a somatic hybrid line derived from Nicotiana tabacum L. and Nicotiana suaveolens Lehm. This mutation is inherited maternally. Young, half-expanded Vir-c leaves contained three to six times less chlorophyll than did control leaves, and reached maximum chlorophyll levels much later in development. Chlorophyll synthesis rates and chloroplast numbers per cell in Vir-c were similar to the control, and carotenoid content in Vir-c was sufficient to protect chlorophyll from photo-oxidation. Photosynthetic rates of Vir-c at low light intensities suggested a reduced ability to collect light. Electron micrographs of Vir-c chloroplasts from half-expanded leaves showed a significant reduction in thylakoids per granum. The decrease in granal thylakoids was strongly associated with low chlorophyll levels; mature Vir-c leaves with nearly normal chlorophyll content showed normal granal profiles. These results are discussed in relation to virescent mutants previously described.

  12. Mutant Resources for the Functional Analysis of the Rice Genome

    National Research Council Canada - National Science Library

    Nili Wang Tuan Long Wen Yao Lizhong Xiong Qifa Zhang Changyin Wu

    2013-01-01

    .... In order to systematically assign functions to all predicted genes in the rice genome, a large number of rice mutant lines, including those created by T-DNA insertion, Ds/dSpm tagging, Tos17 tagging...

  13. Hole poking and motor coordination in lurcher mutant mice.

    Science.gov (United States)

    Lalonde, R; Joyal, C C; Guastavino, J M; Botez, M I

    1993-07-01

    Lurcher mutant mice, a cerebellar mutant displaying ataxia and equilibrium deficits, had fewer hole pokes in a 16-hole matrix than normal mice. Lurcher mutants also took longer to reach a platform from a grid and to begin to climb a grid from the floor. However, the lurchers climbed as high as normal mice on the grid and their exploratory patterns of the holeboard were similar in many respects to normal mice, such as the ratio of center to peripheral hole exploration. In a wooden beam test, although lurchers did not differ from normal mice in terms of the amount of time spent on the beam or in the distance travelled, the mutants were found more often in unstable positions.

  14. Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii.

    Science.gov (United States)

    Sadana, J C; Shewale, J G; Deshpande, M V

    1979-10-01

    A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-beta-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-beta-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.

  15. Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii†

    Science.gov (United States)

    Sadana, J. C.; Shewale, J. G.; Deshpande, M. V.

    1979-01-01

    A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control. Images PMID:16345449

  16. Globulin gene expression in embryos of maize viviparous mutants

    Energy Technology Data Exchange (ETDEWEB)

    Kriz, A.R.; Wallace, M.S.; Paiva, R. (Univ. of Illinois, Urbana (USA))

    1990-02-01

    Expression of genes encoding the major Zea mays embryo globulins was examined in the maize precocious germination viviparous (vp) mutants. Comparison of globulin protein profiles of precociously germinating mutant embryos with those of normally germinating mature embryos revealed substantial differences with respect to the proteins encoded by the Glb1 gene. Analysis of Glb1 transcript levels in vp/vp embryos suggests that these mutants do not fully switch from a program of embryo maturation to one of germination. These preliminary studies indicate that the vp mutants provide an excellent system for the study of embryo maturation in maize. We also provide evidence for the positive regulation of Glb1 expression by the plant growth regulator abscisic acid.

  17. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  18. Characterization of Glutamine-Requiring Mutants of Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Joosten, Han M.L.J.; Herst, Patricia M.; Drift, Chris van der

    1982-01-01

    Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthetase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation

  19. Characterization of Pasteurella multocida mutants of low virulence.

    Science.gov (United States)

    Lee, M D; Glisson, J R; Wooley, R E; Brown, J

    1990-01-01

    Ten temperature-sensitive mutants of the Clemson University (CU) vaccine strain of Pasteurella multocida have been developed and were characterized by phenotypic attributes such as carbohydrate fermentation, antibiotic resistance, and membrane protein profiles. Some mutants were found to have lost the ability to utilize some substrates, notably xylose and gluconate, whereas others were able to ferment additional carbohydrates such as arabinose and rhamnose. CU was found to be resistant to sulfisoxazole, of intermediate resistance to bacitracin, and sensitive to rifampin; the sensitivity to these three antibiotics varied among the mutant strains, but 60% were resistant to rifampin. Membrane protein profiles demonstrated some changes in major bands, and there was variation in 50% of the mutants in proteins in the 31 kilodalton range. All strains were assayed for the presence of several virulence factors, and many were found to produce siderophore and to exhibit some degree of complement resistance.

  20. Selection of mutants of capsicum annuum induced by gamma ray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Y. I.; Lee, Y. B. [Korea Atomic Energy Research Institute, Taejeon (Korea, Republic of); Lee, E. K. [Chungnam National Univ., Taejeon (Korea, Republic of)

    1998-06-01

    For induction and selection of mutations of Capsicum annuum L., dry seeds of pure lines No.1 and No.2 were irradiated with gamma ray of 150Gy, 200Gy and 250Gy. Various mutants were selected such as showing early maturity, short plant height, long fruit and chlorophyll mutations. Mutation frequency of No.1 line was 3.4% in the dose of 150Gy, while the frequency of No.2 line was 2.7% in the dose of 250Gy. For selection of resistant mutant to amino acid analog, the optimum concentration of 5-methyltryptophan (5-MT) and S-(2-aminoethyl)-L-cysteine were 25 ppm and 30 ppm, respectively. Four resistant mutant lines to 5-MT were selected among 400 mutant lines.

  1. Investigating the Structure and Dynamics of the PIK3CA Wild-Type and H1047R Oncogenic Mutant

    Science.gov (United States)

    Pavlaki, Maria; Lazani, Vasiliki; Christoforidis, Savvas; Agianian, Bogos; Cournia, Zoe

    2014-01-01

    The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation. PMID:25340423

  2. phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens.

    Science.gov (United States)

    Elsheimer-Matulova, Marta; Varmuzova, Karolina; Kyrova, Kamila; Havlickova, Hana; Sisak, Frantisek; Rahman, Masudur; Rychlik, Ivan

    2015-09-17

    Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitoring the transcription of 36 genes related to immune response. All the mutants and the wild type strain induced inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1 mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to protective immunity but was associated with inflammation and antibody production. The differences in interaction between the mutants and chicken host can be used for a more detailed understanding of the chicken immune system.

  3. Uv- and Gamma-Radiation Sensitive Mutants of Arabidopsis Thaliana

    OpenAIRE

    Jiang, C Z; Yen, C. N.; Cronin, K; Mitchell, D.; Britt, A B

    1997-01-01

    Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and -independent pathways. The mechanism of the ``dark repair'' pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell in...

  4. [The behavioral development of the mutant "staggerer" mouse].

    Science.gov (United States)

    Guastavino, J M

    1978-01-01

    The behavioural study, in particular rearing environmental conditions, of the mutant mouse staggerer has shown that such animals may live more than 90 days. (he behavioural diagnosis of this mutation has been possible from the second week of life, using specific tests. A typical "bat posture" permits one to recognize the mutant from the normal Mouse. Locomotory and feeding behaviours also present late and various qualitatige particularities.

  5. Structure prediction of subtilisin BPN' mutants using molecular dynamics methods

    OpenAIRE

    Heiner, Andreas P.; Berendsen, Herman J.C.; van Gunsteren, Wilfred F.

    2017-01-01

    In this paper we describe the achievements and pitfalls encountered in doing structure predictions of protein mutants using molecular dynamics simulation techniques in which properties of atoms are slowly changed as a function of time. Basically the method consists of a thermodynamic integration (slow growth) calculation used for free energy determination, but aimed at structure prediction; this allows for a fast determination of the mutant structure. We compared the calculated structure of t...

  6. Fate of peptides in peptidase mutants of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, E.R S; Mierau, I; Poolman, B.; Konings, W.N; Venema, G; Kok, J.

    The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN, pepC, pepO, pepX and pepT were deleted, Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu-Gly-Gly, Gly-Phe-Leu, Leu-Gly-Pro,

  7. Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

    Science.gov (United States)

    Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

    2017-05-01

    An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step.

    Directory of Open Access Journals (Sweden)

    Rebekah N Whitehead

    Full Text Available Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.

  9. Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy.

    Science.gov (United States)

    Yoon, Dennis J; Chen, Kevin Y; Lopes, André M; Pan, April A; Shiloach, Joseph; Mason, Anne B; Kamei, Daniel T

    2017-03-07

    The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole

  10. Biochemical and structural studies of mutants indicate concerted movement of the dimer interface and ligand-binding region of Mycobacterium tuberculosis pantothenate kinase.

    Science.gov (United States)

    Paul, A; Kumar, P; Surolia, A; Vijayan, M

    2017-11-01

    Two point mutants and the corresponding double mutant of Mycobacterium tuberculosis pantothenate kinase have been prepared and biochemically and structurally characterized. The mutants were designed to weaken the affinity of the enzyme for the feedback inhibitor CoA. The mutants exhibit reduced activity, which can be explained in terms of their structures. The crystals of the mutants are not isomorphous to any of the previously analysed crystals of the wild-type enzyme or its complexes. The mycobacterial enzyme and its homologous Escherichia coli enzyme exhibit structural differences in their nucleotide complexes in the dimer interface and the ligand-binding region. In three of the four crystallographically independent mutant molecules the structure is similar to that in the E. coli enzyme. Although the mutants involve changes in the CoA-binding region, the dimer interface and the ligand-binding region move in a concerted manner, an observation which might be important in enzyme action. This work demonstrates that the structure of the mycobacterial enzyme can be transformed into a structure similar to that of the E. coli enzyme through minor perturbations without external influences such as those involving ligand binding.

  11. In vitro exploration of latent prothrombin mutants conveying antithrombin resistance.

    Science.gov (United States)

    Tamura, Shogo; Murata-Kawakami, Moe; Takagi, Yuki; Suzuki, Sachiko; Katsumi, Akira; Takagi, Akira; Kojima, Tetsuhito

    2017-09-20

    Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade. Copyright © 2017. Published by Elsevier Ltd.

  12. NRAS-mutant melanoma: current challenges and future prospect

    Directory of Open Access Journals (Sweden)

    Muñoz-Couselo E

    2017-08-01

    Full Text Available Eva Muñoz-Couselo,1,2 Ester Zamora Adelantado,1,2 Carolina Ortiz,1,2 Jesús Soberino García,3 José Perez-Garcia31Medical Oncology Department, Vall d’Hebron Hospital, Barcelona, Spain; 2Vall d’Hebron Institute of Oncology (VHIO, Barcelona, Spain; 3Baselga Institute of Oncology, Hospital Quirón, Barcelona, SpainAbstract: Melanoma is one of the most common cutaneous cancers worldwide. Activating mutations in RAS oncogenes are found in a third of all human cancers and NRAS mutations are found in 15%–20% of melanomas. The NRAS-mutant subset of melanoma is more aggressive and associated with poorer outcomes, compared to non-NRAS-mutant melanoma. Although immune checkpoint inhibitors and targeted therapies for BRAF-mutant melanoma are transforming the treatment of metastatic melanoma, the ideal treatment for NRAS-mutant melanoma remains unknown. Despite promising preclinical data, current therapies for NRAS-mutant melanoma remain limited, showing a modest increase in progression-free survival but without any benefit in overall survival. Combining MEK inhibitors with agents inhibiting cell cycling and the PI3K–AKT pathway appears to provide additional benefit; in particular, a strategy of MEK inhibition and CDK4/6 inhibition is likely to be a viable treatment option in the future. Patients whose tumors had NRAS mutations had better response to immunotherapy and better outcomes than patients whose tumors had other genetic subtypes, suggesting that immune therapies – especially immune checkpoint inhibitors – may be particularly effective as treatment options for NRAS-mutant melanoma. Improved understanding of NRAS-mutant melanoma will be essential to develop new treatment strategies for this subset of patients with melanoma.Keywords: metastatic melanoma, NRAS mutation, MEK inhibitor, immunotherapy, trametinib, binimetinib

  13. Defective glycinergic synaptic transmission in zebrafish motility mutants

    Directory of Open Access Journals (Sweden)

    Hiromi Hirata

    2010-01-01

    Full Text Available Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

  14. Altered protein dynamics of disease-associated lamin A mutants

    Directory of Open Access Journals (Sweden)

    Worman Howard J

    2004-12-01

    Full Text Available Abstract Background Recent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery. Results We have found that a large proportion of wild-type lamin A at the nuclear periphery is immobile, but that there is some slow movement of lamin A within the nuclear lamina. The mobility of an R482W mutant lamin A was indistinguishable from wild-type, but increased mobility of L85R and L530P mutant proteins within the nuclear lamina was found. However, the N195K mutant shows the most enhanced protein mobility, both within the nucleoplasm and within the lamina. Conclusion The slow kinetics of lamin A movement is compatible with its incorporation into a stable polymer that only exchanges subunits very slowly. All of the myopathy-associated lamin A mutants that we have studied show increased protein movement compared with wild-type. In contrast, the dynamic behaviour of the lipodystrophy-associated lamin A mutant was indistinguishable from wild-type. This supports the hypothesis that the underlying defect in lamin A function is quite distinct in the laminopathies that affect striated muscle, compared to the diseases that affect adipose tissue. Our data are consistent with an alteration in the stability of the lamin A molecules within the higher-order polymer at the nuclear lamina in myopathies.

  15. Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2.

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    Giuseppina Andreotti

    Full Text Available The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG. For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.

  16. PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

    Science.gov (United States)

    Ilic, Nina; Birsoy, Kıvanç; Aguirre, Andrew J.; Kory, Nora; Pacold, Michael E.; Singh, Shambhavi; Moody, Susan E.; DeAngelo, Joseph D.; Spardy, Nicole A.; Freinkman, Elizaveta; Weir, Barbara A.; Cowley, Glenn S.; Root, David E.; Asara, John M.; Vazquez, Francisca; Widlund, Hans R.; Sabatini, David M.; Hahn, William C.

    2017-01-01

    Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate–aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate–aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. PMID:28396387

  17. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

    Science.gov (United States)

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J; Milbrandt, Jeffrey

    2015-07-10

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Development of Bacillus subtilis mutants to produce tryptophan in pigs.

    Science.gov (United States)

    Bjerre, Karin; Cantor, Mette D; Nørgaard, Jan V; Poulsen, Hanne D; Blaabjerg, Karoline; Canibe, Nuria; Jensen, Bent B; Stuer-Lauridsen, Birgitte; Nielsen, Bea; Derkx, Patrick M F

    2017-02-01

    To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l. Genome sequence analysis revealed that M1 had three single nuclear polymorphisms (SNPs) and M2 had two SNPs compared to the wild type strains. In both mutants SNPs were found in genes regulating tryptophan synthesis. Reverse transcription PCR confirmed up-regulation of the tryptophan synthesis genes in both mutants, the expression was up to 3 times higher in M2 than in M1. Tryptophan-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications.

  19. Analysis of AtCry1 and Mutants

    Science.gov (United States)

    Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

    Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

  20. Normal aging modulates the neurotoxicity of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Elsa Diguet

    Full Text Available Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD, a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt, the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week and old (15 month rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

  1. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    Science.gov (United States)

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  2. Structural outline of the detailed mechanism for elongation factor Ts-mediated guanine nucleotide exchange on elongation factor Tu.

    Science.gov (United States)

    Thirup, Søren S; Van, Lan Bich; Nielsen, Tine K; Knudsen, Charlotte R

    2015-07-01

    Translation elongation factor EF-Tu belongs to the superfamily of guanine-nucleotide binding proteins, which play key cellular roles as regulatory switches. All G-proteins require activation via exchange of GDP for GTP to carry out their respective tasks. Often, guanine-nucleotide exchange factors are essential to this process. During translation, EF-Tu:GTP transports aminoacylated tRNA to the ribosome. GTP is hydrolyzed during this process, and subsequent reactivation of EF-Tu is catalyzed by EF-Ts. The reaction path of guanine-nucleotide exchange is structurally poorly defined for EF-Tu and EF-Ts. We have determined the crystal structures of the following reaction intermediates: two structures of EF-Tu:GDP:EF-Ts (2.2 and 1.8Å resolution), EF-Tu:PO4:EF-Ts (1.9Å resolution), EF-Tu:GDPNP:EF-Ts (2.2Å resolution) and EF-Tu:GDPNP:pulvomycin:Mg(2+):EF-Ts (3.5Å resolution). These structures provide snapshots throughout the entire exchange reaction and suggest a mechanism for the release of EF-Tu in its GTP conformation. An inferred sequence of events during the exchange reaction is presented. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Scale up of dextran production from a mutant of Pediococcus pentosaceus (SPAm using optimized medium in a bioreactor

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    Seema Patel

    2011-12-01

    Full Text Available The mutant of Pediococcus pentosaceus (SPAm produced earlier by UV-mutagenesis exhibiting higher dextransucrase activity as compared to wild-type was used. The generated mutant SPAm gave 12.2 mg/ml, a 20% higher dextran than wild-type. Response surface methodology was carried out for further enhancement of dextran production. To enhance dextran production by the mutant SPAm, Plackett-Burman Design and a 2² full factorial Central Composite Design was employed. After response optimization, the optimum concentration of sucrose and yeast extract was 5.115% (w/v and 0.635% (w/v, respectively. The experimental values of dextran 36.0 mg/ml at flask level and 35.0 mg/ml at bioreactor level were in good agreement with the predicted value of 40.8 mg/ml. The increase in dextran production by the mutant SPAm using the optimized medium was 3 fold higher as compared to unoptimized medium.

  4. Generation and Characterization of dickkopf3 Mutant Mice

    Science.gov (United States)

    del Barco Barrantes, Ivan; Montero-Pedrazuela, Ana; Guadaño-Ferraz, Ana; Obregon, Maria-Jesus; Martinez de Mena, Raquel; Gailus-Durner, Valérie; Fuchs, Helmut; Franz, Tobias J.; Kalaydjiev, Svetoslav; Klempt, Martina; Hölter, Sabine; Rathkolb, Birgit; Reinhard, Claudia; Morreale de Escobar, Gabriella; Bernal, Juan; Busch, Dirk H.; Wurst, Wolfgang; Wolf, Eckhard; Schulz, Holger; Shtrom, Svetlana; Greiner, Erich; Hrabé de Angelis, Martin; Westphal, Heiner; Niehrs, Christof

    2006-01-01

    dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity. PMID:16508007

  5. Some enzyme activities of acetate mutants of Yarrowia lypolytica

    Energy Technology Data Exchange (ETDEWEB)

    Robak, M.; Wojtatowicz, M.; Rymowicz, W. [Akademia Rolnicza, Wroclaw (Poland)

    1994-12-31

    Activity at the following enzymes: CS (oxaloacetate-lyase citrate), AH (citrate (isocitrate) hydrolyase), ICDH (threo-Ds-isocitrate: NADP oxidoreductase) and ICL (threo-Ds-isocitrate glyoxyglate-lyase) was measured at subsequent stages of citrate fermentation on glucose by wild type strain `Y, lipolytica A-101` and 2 acetate defective mutants, in order to recognize metabolic disorders in those mutants, which resulted in markedly improved homogeneity of citric acid production. Mutants did not show significant changes in activity of TCA cycle enzymes and ICL. Thus suggests that the control of citric:isocitric acid ratio is more difficult and it can also depend on transportation systems of both acids. (author). 19 refs, 3 figs, 2 tabs.

  6. The swimming activity of the staggerer mutant mouse.

    Science.gov (United States)

    Goodall, G; Guastavino, J M; Gheusi, G

    1986-09-01

    Four experiments investigated the swimming behaviour of staggerer mutant mice. The results partially confirmed previous reports that a mouse's swimming is unaffected by the staggerer mutation. In terms of speed and distance there are indeed no measurable differences between normal and staggerer mice, when first placed in the water. The stagger's resistance was however shown to be much lower than a normal's and the genetic difference was also associated with different styles of swimming. Furthermore, whereas the normal mouse's swimming behaviour evolves with increased time in the water, the staggerer's remains constant. The differences are interpreted on the basis of abnormal novelty reactions by the staggerer mutants. Thus, swimming appears to be a better tool for investigating the higher-level cognitive functions of this mutant than terrestrial locomotion. Copyright © 1986. Published by Elsevier B.V.

  7. Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Lalonde, R; Strazielle, C

    2011-04-15

    Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage. Copyright © 2010. Published by Elsevier B.V.

  8. Cellular differentiation regulated by gibberellin in the Arabidopsis thaliana pickle mutant

    Energy Technology Data Exchange (ETDEWEB)

    Ogas, J.; Somerville, C. [Carnegie Institution of Washington, Stanford, CA (United States); Cheng, Jin-Chen; Sung, R. [Univ. of California, Berkeley, CA (United States)

    1997-07-04

    The plant growth regulator gibberellin (GA) has a profound effect on shoot development and promotes developmental transitions such as flowering. Little is known about any analogous effect GA might have on root development. In a screen for mutants, Arabi-dopsis plants carrying a mutation designated pickle (pkl) were isolated in which the primary root meristem retained characteristics of embryonic tissue. Expression of this aberrant differentiation state was suppressed by GA. Root tissue from plants carrying the pkl mutation spontaneously regenerated new embryos and plants. 19 refs., 3 figs., 1 tab.

  9. Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

    Directory of Open Access Journals (Sweden)

    Edson Jiovany Ramírez-Nava

    2017-10-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD and G6PD Nefza (Leu323Pro, and the double mutant G6PD A− (Asn126Asp + Leu323Pro. The mutants showed lower residual activity (≤50% of WT G6PD and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

  10. Genetic interactions among homologous recombination mutants in Candida albicans.

    Science.gov (United States)

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Comparison Study on Colonization of hilA Mutant and Parent Strains of Salmonella enteritidis in Vertically Infected Broiler Chickens

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    MohammadSadegh Madadi

    2015-10-01

    Full Text Available Background: Salmonella actively stimulates its own uptake into the epithelial cells by inducing cytoskeleton rearrangements and membrane ruffling triggered by some proteins secreted by Salmonella into the cytosol of the epithelial cells via a type III secretion system (TTSS encoded bygenes of the Salmonella pathogenicity island 1 (SPI-1. hilA is a transcriptional activator encoded on Salmonella Pathogenicity Island 1 (SPI-1 genes.Methods: To assess the importance of hilA in a simulation modeling of vertical infection and shedding of S. enteritidis in broiler chickens a long-term experiment was designed. Two groups of 200 fertile eggs were inoculated with 20 colony forming units (CFU of hilA mutant of S. enteritidis or its parent strain just prior to incubation. Thirty five birds of each group were housed in separate rooms. On days 2, 4, 7, 14, 21, 28 and 35 of age, cloacal swabs from live birds as well as samples from internal organs (intestinal tract, liver and spleen were evaluated by bacteriological or molecular methods.Results: In most of sampling days colonization and invasion of parent strain S. enteritidis in intestine (especially ceaca and internal organs of chickens were higher with compared to its hilA mutant but this mutant strain could still colonize in intestinal tract and even invade liver or spleen.Conclusion: Colonization of hilA mutant of S. enteritidis indicated that hilA gene is only one part of the modulators in Salmonella invasion mechanism. The ability of hilA mutant to multiply and persist in host internal organs including ceaca may promise further research for potential of hilA mutant to prevent the initial colonization of the intestinal tract by a virulent S. enteritidis strain

  12. RFLP mapping of the barley homeotic mutant lax-a.

    Science.gov (United States)

    Laurie, D A; Pratchett, N; Allen, R L; Hantke, S S

    1996-07-01

    The lax-a homeotic mutant of barley has flowers in which lodicules are replaced by stamens (giving five stamens per flower). RFLP mapping of an F2 population from a Bonus lax-a (1) x H. spontaneum cross showed that the mutation was on the short arm of chromosome 7(5H), closely linked to the centromere. An additional F2 population was used to show that the lax-a mutation gave the five-stamen phenotype in all flowers of 6-rowed spikes and that hoods were elevated and reduced in size in lax-a/Hooded double-mutant plants.

  13. Mutant E. coli strain with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  14. Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

    DEFF Research Database (Denmark)

    Fukai, Eigo; Malolepszy, Anna; Sandal, Niels Nørgaard

    2014-01-01

    The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next......-generation sequencing approaches. The LORE1 mutant lines are freely available and can be ordered online. Endogenous retrotransposons are also active in many other plant species. Based on the methods developed for LORE1 mutagenesis, it should be simple to establish similar systems in other species, once an appropriate...

  15. Mutant E. coli strain with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  16. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  17. Clear Plaque Mutants of Lactococcal Phage TP901-1

    DEFF Research Database (Denmark)

    Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome...... protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1....

  18. A male sterile pepper (C. annuum L.) mutant.

    Science.gov (United States)

    Daskaloff, S

    1968-08-01

    1. After treatment of dry seeds of red pepperCapsicum annuum L. with X-rays a male-sterile mutant was discovered in the M2. 2. The male-sterile mutant segregates in a ratio of 3.28:1 (χ(2)=3.148, probability 0.07). 3. After an alternative cultivation of male-sterile plants and of a variety with good combining ability relatively good fruit-setting and seed production was obtained. 4. Grafting of male-sterile scions to normal stocks does not affect the male-sterile phenotype.

  19. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    Science.gov (United States)

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  20. Effects of salt stress on wild type and vte4 mutant Arabidopsis thaliana: Model plant to engineer tolerance towards salinity

    Directory of Open Access Journals (Sweden)

    Khalatbari Amir Ali

    2013-01-01

    Full Text Available One of the major environmental constraints impairing plant distribution and yield is believed to be salt stress. Additionally, engineered abiotic stress resistance or/and tolerance is considered as an indispensable target in order to enhance plant productivity. In this study, the effects of salinity on physiological and morphological of wild type (Columbia-0 and vte4 mutant Arabidopsis thaliana were investigated under different NaCl concentrations. These salt treatments, including control condition, 50mM and 100mM NaCl were imposed on the plants. Each salt treatment was replicated three times in a complete randomized design with factorial arrangement. Wild type and mutant A.thaliana plants were subjected to the abiotic stress (salinity for up to 11 days to evaluate the parameters of growth, development and water relations. As a result, the performance of wild type plants was stronger than vte4 mutant under different salt treatments. Under control condition, rosette dry weight, maximum quantum efficiency (PSII and specific leaf area obtained the highest values of 13.85 mg, considered, wild type A.thaliana recorded higher value of 0.82 gW/gFW for relative water content (RWC under 50mM NaCl whereas mutant plants gained the value of 0.78 gW/gFW under the same condition. However, root mass fraction indicated an increase for both wild type and vte4 mutant plants after 11 days of salt stress onset. The reduction of water potential was observed for wild type and mutant A.thaliana where it scored -1.3 MPa and -1.4, respectively. As a conclusion, these findings implied that under different salt treatments morphological and physiological responses of wild type and vte4 mutant were affected in which wild type plants showed more tolerance. Lack of γ-tocopherol methyltransferase (γ -TMT gene in vte4 seemed to impair defence mechanism of this mutant against salinity.

  1. Mutant connexin 50 (S276F) inhibits channel and hemichannel ...

    Indian Academy of Sciences (India)

    The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly ... Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Donghu Road 169#, Wuhan, Hubei 430071, People's Republic of China; Hubei Cancer ...

  2. Locating a modifier gene of Ovum mutant through crosses between ...

    Indian Academy of Sciences (India)

    RESEARCH ARTICLE Volume 95 Issue 2 June 2016 pp 297-302 ... mouse; DDK syndrome; ovum mutant; modifier gene; quantitative trait loci ... Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other ...

  3. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ARL

    mutant, the embryo sac becomes smaller and bulbous, and ultrastructure of developing cotyledons exhibits ... has higher protein and oil content, and has altered seed ultrastructure. The purpose of this experiment is to evaluate the features of soybean curled-cotyledons ..... mitochondria of soybean seedling cotyledons.

  4. Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus

    NARCIS (Netherlands)

    Oklejewicz, Malgorzata; Daan, Serge

    The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span

  5. Molecular analysis of sex chromosome-linked mutants in the ...

    Indian Academy of Sciences (India)

    2010-09-06

    Sep 6, 2010 ... In B. mori, polyploid individuals with different sex chromo- some constitutions can be ... RNA was extracted from the ovary, testis, and fat body of a fifth-instar larva, and from the an- tennae of 10 moths. The primer set .... For example, the body of sk. (stick, 4–25.8) mutant larvae is firm to the touch, while that.

  6. Characterization of resistant tomato mutants to bacterial canker ...

    African Journals Online (AJOL)

    Yomi

    2012-04-19

    Apr 19, 2012 ... A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the ...

  7. Growth properties of Cellulomonas flavigena mutants affected in cellulose utilization.

    Science.gov (United States)

    Béguin, P; Eisen, H

    1978-01-01

    The role of cellobiose metabolism in cellulose utilization by Cellulomonas flavigena was investigated by studying mutants unable to grow on cellobiose or cellulose. The results show that the ability to utilize cellulose is strictly dependent on the ability to utilize cellobiose. PMID:415038

  8. Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

    DEFF Research Database (Denmark)

    Jensen, Anders Bøgh; Raventos, D.; Mundy, John Williams

    2002-01-01

    Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis. To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC...

  9. Molecular analysis of mutants of the Neurospora adenylosuccinate ...

    Indian Academy of Sciences (India)

    2012-08-07

    Aug 7, 2012 ... Abstract. The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well- characterized N. crassa ad-8 mutants in the ...

  10. Performance of early maturing mutants derived from 'supa' rice ...

    African Journals Online (AJOL)

    Days to 50% flowering and 1000 grain weight exerted negative direct effect on yield. Changes in grain quality were also observed emphasizing the importance of conducting cooking and taste panel tests. Keywords: Early maturity, grain guality, rice mutants, Oryza saliva, path coefficient. Tanzania J. Agri. Sc. (2001) Vol 4, ...

  11. Isolation and partial characterization of carotenoid mutants of ...

    African Journals Online (AJOL)

    Global Journal of Pure and Applied Sciences ... Three major pigments isolated by high performance liquid chromatography (HPLC) were characterized by their absorption maxima, partition ratios in light ... High performance liquid chromatography was used to compare pigments of the wild-type with those of the mutants.

  12. Dynamics of Mutant Cells in Hierarchical Organized Tissues

    Science.gov (United States)

    Werner, Benjamin; Dingli, David; Lenaerts, Tom; Pacheco, Jorge M.; Traulsen, Arne

    2011-01-01

    Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance. PMID:22144884

  13. Characterization of mutant cowpea [ Vigna unguiculata (L) Walp ...

    African Journals Online (AJOL)

    Phylogenetic relationship and polymorphism was detected in 10 cowpea lines comprising of leaf, flower and stem mutants, their putative parents and an exotic accession using 10 random ... Genetic distance ranged from 0.05 to 0.30 based on AFLP markers, while it ranged between 0.13 and 0.44 for RAPD markers. Cluster ...

  14. Clustering common bean mutants based on heterotic groupings ...

    African Journals Online (AJOL)

    The objective of this study was to cluster bean mutants from a bean mutation breeding programme, based on heterotic groupings. This was achieved by genotyping 16 bean genotypes, using 21 Simple Sequence Repeats (SSR) bean markers. From the results, three different clusters A, B and C, were obtained suggesting ...

  15. Insulator dysfunction and oncogene activation in IDH mutant gliomas.

    Science.gov (United States)

    Flavahan, William A; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Venteicher, Andrew S; Stemmer-Rachamimov, Anat O; Suvà, Mario L; Bernstein, Bradley E

    2016-01-07

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

  16. IGFBP2 expression predicts IDH-mutant glioma patient survival.

    Science.gov (United States)

    Huang, Lin Eric; Cohen, Adam L; Colman, Howard; Jensen, Randy L; Fults, Daniel W; Couldwell, William T

    2017-01-03

    Mutations of the isocitrate dehydrogenase (IDH) 1 and 2 genes occur in ~80% of lower-grade (WHO grade II and grade III) gliomas. Mutant IDH produces (R)-2-hydroxyglutarate, which induces DNA hypermethylation and presumably drives tumorigenesis. Interestingly, IDH mutations are associated with improved survival in glioma patients, but the underlying mechanism for the difference in survival remains unclear. Through comparative analyses of 286 cases of IDH-wildtype and IDH-mutant lower-grade glioma from a TCGA data set, we report that IDH-mutant gliomas have increased expression of tumor-suppressor genes (NF1, PTEN, and PIK3R1) and decreased expression of oncogenes(AKT2, ARAF, ERBB2, FGFR3, and PDGFRB) and glioma progression genes (FOXM1, IGFBP2, and WWTR1) compared with IDH-wildtype gliomas. Furthermore, each of these genes is prognostic in overall gliomas; however, within the IDH-mutant group, none remains prognostic except IGFBP2 (encodinginsulin-like growth factor binding protein 2). Through validation in an independent cohort, we show that patients with low IGFBP2 expressiondisplay a clear advantage in overall and disease-free survival, whereas those with high IGFBP2 expressionhave worse median survival than IDH-wildtype patients. These observations hold true across different histological and molecular subtypes of lower-grade glioma. We propose therefore that an unexpected biological consequence of IDH mutations in glioma is to ameliorate patient survival by promoting tumor-suppressor signaling while inhibiting that of oncogenes, particularly IGFBP2.

  17. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    Science.gov (United States)

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  18. Development and evaluation of drought resistant mutant germ ...

    African Journals Online (AJOL)

    terms of relative water content, free proline concentration and yield. The yield ... are suitable for boiling and canning. .... definition the permanent wilting point is the soil water content at which plants do not recover turgor overnight, but will recover if water is applied. Selected mutant and control plants were planted in pots in a.

  19. Complementation of sweet corn mutants: a method for grouping ...

    Indian Academy of Sciences (India)

    accumulate sugars at the expense of starch and have low total carbohydrate at the mature kernel stage (Boyer and. Shannon 1984). At 18–21 days after pollination (harvest stage of sweet corn), these mutants have four to eight times higher total sugar than the normal corn (Holder et al. 1974). Due to comparative high sugar ...

  20. Characterization of resistant tomato mutants to bacterial canker ...

    African Journals Online (AJOL)

    A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the constructed M2 population, ...

  1. A dwarf wheat mutant is associated with increased drought ...

    African Journals Online (AJOL)

    Owner

    'Green revolution' genes encode mutant gibberellin response modulators. Nature 400 (6741):. 256-61. Zhang et al. 1057. Peng J, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP,. Harberd NP (1997). The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses Genes Dev.

  2. Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy.

    Directory of Open Access Journals (Sweden)

    Xin Dai

    Full Text Available KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors.

  3. Modelling the evolution and spread of HIV immune escape mutants.

    Science.gov (United States)

    Fryer, Helen R; Frater, John; Duda, Anna; Roberts, Mick G; Phillips, Rodney E; McLean, Angela R

    2010-11-18

    During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level.

  4. Enhanced sporulation and toxin production by a mutant derivative of ...

    African Journals Online (AJOL)

    fatima

    based medium. Maximum spore and crystal proteins were produced at 40°C with corn steep liquor as nitrogen source and hydrol as a carbon source. The best mutant MUV7 supported .... containing 10 L of culture medium as described earlier (Ghribi et al.,. 2004). ..... Saccharomyces cerevisiae ITV strain (Ortiz-Muniz et al.,.

  5. nitrosoguanidine-induced cadmium resistant mutants of Aspergillus ...

    Indian Academy of Sciences (India)

    Unknown

    of S. cerevisiae (Inouhe et al 1989) were used for under- standing the molecular genetics of cadmium toxicity and ... The pH of the medium was adjusted to 6⋅4 before autoclaving. Cadmium-resistant mutants after isolation ..... Saccharomyces cerevisiae; Biochem. Biophys. Acta 993 51–55. Kimura M, Otaki N and Imano M ...

  6. Development of a mutant strain of Bacillus subtilis showing ...

    African Journals Online (AJOL)

    Through fermentation experiments, it was confirmed that the mutant strain, TH-49, was not capable of using acetoin accumulated in broth as its energy sources for growth after glucose was consumed. This phenomenon was inconsistent with that the majorities of bacteria accumulate acetoin as stored energy sources and ...

  7. Transcriptional Analysis of serk1 and serk3 coreceptor mutants

    NARCIS (Netherlands)

    Esse, van Wilma; Hove, ten Colette A.; Guzzonato, Francesco; Esse, van Peter; Boekschoten, Mark; Ridder, Lars; Vervoort, Jacques; Vries, de Sacco C.

    2016-01-01

    Somatic embryogenesis receptor kinases (SERKs) are ligand-binding coreceptors that are able to combine with different ligandperceiving receptors such as BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2. Phenotypical analysis of serk single mutants is not straightforward because

  8. Screening of allyl alcohol resistant mutant of Rhizopus oryzae and ...

    African Journals Online (AJOL)

    Ethanol is a main by-product in the fermentation broth of Rhizopus oryzae during the production of high-optical purity L-lactic acid. By screening the lower activity of alcohol dehydrogenase (ADH) mutant, thus decreasing the flux of pyruvic acid to ethanol may be a virtual method for increasing the conversion rate of glucose ...

  9. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. A dwarf wheat mutant is associated with increased drought ...

    African Journals Online (AJOL)

    ... was significantly higher than Jingdong 6. Most of the s-dwarf seedlings survived in recovering experiement after water loss. The stalk of s-dwarf seedling also showed reduced gravitropism. This is the first report about a new dwarf wheat mutant associated with increased drought resistance and altered stalk gravitropism.

  11. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila

    Indian Academy of Sciences (India)

    2014-07-20

    Jul 20, 2014 ... from the mutation. As a result, Pdf01 mutant flies locomote with precise rhythmicity generated by one ultradian oscilla- tor. In future studies, we would like to dissect this system using genetic tools available in Drosophila, for example by silencing a particular set of Pdf-positive neurons. Circadian rhythms are ...

  12. Dynamics of mutant cells in hierarchical organized tissues.

    Directory of Open Access Journals (Sweden)

    Benjamin Werner

    2011-12-01

    Full Text Available Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance.

  13. Siim Nestor soovitab : Mutant Disco. Azymuth. Klubis Hollywood / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2003-01-01

    Mutant Disco klubis Prive 4. juulil. Brasiilia jazz-trio Azmuth klubis BonBon 5. juulil. Pidustuste sarja Hip Hop Cafe sünnipäeva tähistamisest klubis Hollywood 4. juulil, üritusest Ibiza Night 5. juulil

  14. Photophysics and optical switching in green fluorescent protein mutants

    NARCIS (Netherlands)

    Creemers, T.M.H.; Lock, A.J.; Subramaniam, V.; Jovin, T.M.; Völker, S.

    2000-01-01

    We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo- interconverted among three conformations and are, therefore, not photostable 'one-color' systems as previously believed. From our experiments we have further derived

  15. Genetic characterization of glossy-leafed mutant broccoli lines

    Science.gov (United States)

    Glossy mutants of Brassica oleracea L. have reduced or altered epicuticular wax on the surface of their leaves as compared to wild-type plants, conveying a shiny green appearance. Mutations conferring glossiness are common and have been found in most B. oleracea crop varieties, including cauliflower...

  16. Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

    Science.gov (United States)

    Matsuura, Yoshinori; Takehira, Michiyo; Joti, Yasumasa; Ogasahara, Kyoko; Tanaka, Tomoyuki; Ono, Naoko; Kunishima, Naoki; Yutani, Katsuhide

    2015-10-26

    Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

  17. Mutant p53 as a target for cancer treatment.

    Science.gov (United States)

    Duffy, Michael J; Synnott, Naoise C; Crown, John

    2017-09-01

    TP53 (p53) is the single most frequently altered gene in human cancers, with mutations being present in approximately 50% of all invasive tumours. However, in some of the most difficult-to-treat cancers such as high-grade serous ovarian cancers, triple-negative breast cancers, oesophageal cancers, small-cell lung cancers and squamous cell lung cancers, p53 is mutated in at least 80% of samples. Clearly, therefore, mutant p53 protein is an important candidate target against which new anticancer treatments could be developed. Although traditionally regarded as undruggable, several compounds such as p53 reactivation and induction of massive apoptosis-1 (PRIMA-1), a methylated derivative and structural analogue of PRIMA-1, i.e. APR-246, 2-sulfonylpyrimidines such as PK11007, pyrazoles such as PK7088, zinc metallochaperone-1 (ZMC1), a third generation thiosemicarbazone developed by Critical Outcome Techonologies Inc. (COTI-2) as well as specific peptides have recently been reported to reactive mutant p53 protein by converting it to a form exhibiting wild-type properties. Consistent with the reactivation of mutant p53, these compounds have been shown to exhibit anticancer activity in preclinical models expressing mutant p53. To date, two of these compounds, i.e. APR-246 and COTI-2 have progressed to clinical trials. A phase I/IIa clinical trial with APR-246 reported no major adverse effect. Currently, APR-246 is undergoing a phase Ib/II trial in patients with advanced serous ovarian cancer, while COTI-2 is being evaluated in a phase I trial in patients with advanced gynaecological cancers. It remains to be shown however, whether any mutant p53 reactivating compound has efficacy for the treatment of human cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

    Science.gov (United States)

    Chachoua, Ilyas; Pecquet, Christian; El-Khoury, Mira; Nivarthi, Harini; Albu, Roxana-Irina; Marty, Caroline; Gryshkova, Vitalina; Defour, Jean-Philippe; Vertenoeil, Gaëlle; Ngo, Anna; Koay, Ann; Raslova, Hana; Courtoy, Pierre J; Choong, Meng Ling; Plo, Isabelle; Vainchenker, William; Kralovics, Robert; Constantinescu, Stefan N

    2016-03-10

    Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling. © 2016 by The American Society of Hematology.

  19. Metabolic reprogramming in mutant IDH1 glioma cells.

    Directory of Open Access Journals (Sweden)

    Jose L Izquierdo-Garcia

    Full Text Available Mutations in isocitrate dehydrogenase (IDH 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS-detectable changes in the cellular metabolome.Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data.Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

  20. Elevation of Urinary 2-Hydroxyglutarate in IDH-Mutant Glioma.

    Science.gov (United States)

    Fathi, Amir T; Nahed, Brian V; Wander, Seth A; Iafrate, A John; Borger, Darrell R; Hu, Ranliang; Thabet, Ashraf; Cahill, Daniel P; Perry, Ashley M; Joseph, Christelle P; Muzikansky, Alona; Chi, Andrew S

    2016-02-01

    Recurrent mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, which are frequent in gliomas, result in marked accumulation of the metabolic by-product 2-hydroxyglutarate (2-HG) within tumors. In other malignancies, such as acute myeloid leukemia, presence of IDH mutation is associated with elevated 2-HG levels in serum or urine compartments. Circulating 2-HG in patients with glial malignancies has not been thoroughly investigated. In this study, we analyzed 2-HG levels in the serum and urine of a large set of patients with IDH-mutant and IDH-wild-type glioma, and the cerebrospinal fluid (CSF) from a subset of this cohort. We found that 2-HG was elevated in the urine of patients with IDH-mutant versus IDH-wild-type glioma, although no significant differences in 2-HG levels were observed in the serum or the small set of CSF samples obtained. Among patients with IDH-mutant glioma, 2-HG levels did not differ based on the histopathologic grade, genetic subtype (TP53 mutant or 1p/19q codeleted), presence of a canonical (IDH1 R132H) or noncanonical (any other IDH variant) mutation, or treatment type. Our finding suggests that urinary 2-HG is increased among patients with IDH-mutant gliomas, and may represent a future surrogate, noninvasive biomarker to aid in diagnosis, prognosis, and management. Patients with glioma who harbor mutations in isocitrate dehydrogenase genes showed selective elevation of the oncometabolite 2-hydroxyglutarate in the urine. Similar elevations were not identified in the serum or cerebrospinal fluid. 2-Hydroxyglutarate may serve as a useful, noninvasive biomarker to stratify patients newly diagnosed with glioma with regard to prognosis and management. ©AlphaMed Press.

  1. Arabidopsis mutant bik1 exhibits strong resistance to Plasmodiophora brassicae

    Directory of Open Access Journals (Sweden)

    Tao Chen

    2016-09-01

    Full Text Available Botrytis-induced kinase1 (BIK1, a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1 and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2 and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2 and npr1-1 (non-expresser of PR genes that regulate systemic acquired resistance (SAR mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms enhance the resistance to clubroot disease.

  2. The Activity of Nodules of the Supernodulating Mutant Mtsunn Is not Limited by Photosynthesis under Optimal Growth Conditions

    Science.gov (United States)

    Cabeza, Ricardo A.; Lingner, Annika; Liese, Rebecca; Sulieman, Saad; Senbayram, Mehmet; Tränkner, Merle; Dittert, Klaus; Schulze, Joachim

    2014-01-01

    Legumes match the nodule number to the N demand of the plant. When a mutation in the regulatory mechanism deprives the plant of that ability, an excessive number of nodules are formed. These mutants show low productivity in the fields, mainly due to the high carbon burden caused through the necessity to supply numerous nodules. The objective of this study was to clarify whether through optimal conditions for growth and CO2 assimilation a higher nodule activity of a supernodulating mutant of Medicago truncatula (M. truncatula) can be induced. Several experimental approaches reveal that under the conditions of our experiments, the nitrogen fixation of the supernodulating mutant, designated as sunn (super numeric nodules), was not limited by photosynthesis. Higher specific nitrogen fixation activity could not be induced through short- or long-term increases in CO2 assimilation around shoots. Furthermore, a whole plant P depletion induced a decline in nitrogen fixation, however this decline did not occur significantly earlier in sunn plants, nor was it more intense compared to the wild-type. However, a distinctly different pattern of nitrogen fixation during the day/night cycles of the experiment indicates that the control of N2 fixing activity of the large number of nodules is an additional problem for the productivity of supernodulating mutants. PMID:24727372

  3. Kindlin-1 mutant zebrafish as an in vivo model system to study adhesion mechanisms in the epidermis.

    Science.gov (United States)

    Postel, Ruben; Margadant, Coert; Fischer, Boris; Kreft, Maaike; Janssen, Hans; Secades, Pablo; Zambruno, Giovanna; Sonnenberg, Arnoud

    2013-09-01

    From a forward genetic screen for epidermal defects in zebrafish, we identified a loss-of-function mutation in Kindlin-1, an essential regulator of integrin function. The mutation generates a premature stop codon, deleting the integrin-binding site. The mutant zebrafish develops cell-matrix and cell-cell adhesion defects in the basal epidermis leading to progressive fin rupturing, and was therefore designated rupturing-of-fins (rof). Similar defects were observed in the epidermis of Kindler syndrome patients, carrying a loss-of-function mutation in kindlin-1. Mutational analysis and rescue experiments in zebrafish revealed that residues K610, W612, and I647 in the F3 domain are essential for Kindlin-1 function in vivo, and that Kindlin-2 can functionally compensate for the loss of Kindlin-1. The fin phenotype of rof/kindlin-1 mutants resembles that of badfin mutants, carrying a mutation in integrin α3. We show here that this mutation impairs the biosynthesis of integrin α3β1 and causes cell-matrix and cell-cell defects in vivo. Whereas both Integrin-linked kinase (Ilk) and Kindlin-1 cooperate with Integrin α3β1 to resist trauma-induced epidermal defects, Kindlin-1 and Ilk, surprisingly, do not act synergistically but in parallel. Thus, the rof/kindlin-1 mutant zebrafish provides a unique model system to study epidermal adhesion mechanisms in vivo.

  4. The Activity of Nodules of the Supernodulating Mutant Mtsunn Is not Limited by Photosynthesis under Optimal Growth Conditions

    Directory of Open Access Journals (Sweden)

    Ricardo A. Cabeza

    2014-04-01

    Full Text Available Legumes match the nodule number to the N demand of the plant. When a mutation in the regulatory mechanism deprives the plant of that ability, an excessive number of nodules are formed. These mutants show low productivity in the fields, mainly due to the high carbon burden caused through the necessity to supply numerous nodules. The objective of this study was to clarify whether through optimal conditions for growth and CO2 assimilation a higher nodule activity of a supernodulating mutant of Medicago truncatula (M. truncatula can be induced. Several experimental approaches reveal that under the conditions of our experiments, the nitrogen fixation of the supernodulating mutant, designated as sunn (super numeric nodules, was not limited by photosynthesis. Higher specific nitrogen fixation activity could not be induced through short- or long-term increases in CO2 assimilation around shoots. Furthermore, a whole plant P depletion induced a decline in nitrogen fixation, however this decline did not occur significantly earlier in sunn plants, nor was it more intense compared to the wild-type. However, a distinctly different pattern of nitrogen fixation during the day/night cycles of the experiment indicates that the control of N2 fixing activity of the large number of nodules is an additional problem for the productivity of supernodulating mutants.

  5. The Impact of Collisions on the Ability to Detect Rare Mutant Alleles Using Barcode-Type Next-Generation Sequencing Techniques

    Directory of Open Access Journals (Sweden)

    Jenna VanLiere Canzoniero

    2017-07-01

    Full Text Available Barcoding techniques are used to reduce error from next-generation sequencing, with applications ranging from understanding tumor subclone populations to detecting circulating tumor DNA. Collisions occur when more than one sample molecule is tagged by the same unique identifier (UID and can result in failure to detect very-low-frequency mutations and error in estimating mutation frequency. Here, we created computer models of barcoding technique, with and without amplification bias introduced by the UID, and analyzed the effect of collisions for a range of mutant allele frequencies (1e−6 to 0.2, number of sample molecules (10 000 to 1e7, and number of UIDs (4 10 -4 14 . Inability to detect rare mutant alleles occurred in 0% to 100% of simulations, depending on collisions and number of mutant molecules. Collisions also introduced error in estimating mutant allele frequency resulting in underestimation of minor allele frequency. Incorporating an understanding of the effect of collisions into experimental design can allow for optimization of the number of sample molecules and number of UIDs to minimize the negative impact on rare mutant detection and mutant frequency estimation.

  6. Optimization Studies for Enhancing Cellulase Production by Penicillium janthinellum Mutant EU2D-21 Using Response Surface Methodology

    OpenAIRE

    Anil Kumar Nagraj; Mamata Singhvi; Ravi Kumar, V.; Digambar Gokhale

    2014-01-01

    Extracellular fungal cellulases are key enzymes for the degradation of lignocellulosic biomass. Greater production of these enzymes could reduce the cost of biofuels production. In this study, the basal medium for cellulase production by a Penicillium janthinellum mutant (EU2D-21) in submerged fermentation conditions was optimized using response surface methodology (RSM). Initial studies using a Plackett-Burman design (PBD) showed that (NH4)2SO4 and urea are significant factors for improving ...

  7. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

    Science.gov (United States)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

  8. Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; McKinney, Lea V; Pike, Helen

    2008-01-01

    The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11...

  9. Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant.

    Science.gov (United States)

    Silber, J; Bazin, C; Le Menn, A

    1989-09-01

    Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vgB/+ flies are reared on these substances (vgB is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies. Our data suggest that the vg+ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.

  10. Efficient transduction of LEDGF/p75 mutant cells by complementary gain-of-function HIV-1 integrase mutant viruses

    Directory of Open Access Journals (Sweden)

    Hao Wang

    2014-01-01

    Full Text Available Controlling the specificity of retroviral DNA integration could improve the safety of gene therapy vectors, and fusions of heterologous chromatin binding modules to the integrase (IN–binding domain from the lentiviral integration host cofactor lens epithelium–derived growth factor (LEDGF/p75 are a promising retargeting strategy. We previously proposed the utility of IN mutant lentiviral vectors that are selectively activated by complementary LEDGF/p75 variants, and our initial modifications in human immunodeficiency virus type 1 IN and LEDGF/p75 supported about 13% of wild-type vector transduction activity. Here we describe the selection and characterization of the K42E gain-of-function mutation in IN, which greatly improves the efficiency of this system. Both K42E and initial reverse-charge mutations in IN negatively affected reverse transcription and integration, yet when combined together boosted viral transduction efficiency to ∼75% of the wild-type vector in a manner dependent on a complementary LEDGF/p75 variant. Although the K42E mutation conferred functional gains to IN mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant. We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge IN mutant viral and LEDGF/p75 host proteins.

  11. Screening of Metarhizium anisopliae UV-induced mutants for faster growth yields a hyper-virulent isolate with greater UV and thermal tolerances.

    Science.gov (United States)

    Zhao, Jing; Yao, Ruina; Wei, Yun; Huang, Song; Keyhani, Nemat O; Huang, Zhen

    2016-11-01

    The insect pathogenic fungus Metarhizium anisopliae is an important insect biological control agent commercialized for use worldwide. Fungal infection is percutaneous, and rapid germination and growth has been linked to virulence. Using a simple in vitro growth screen to isolate mutants with increased virulence, M. anisopliae SM04 conidia were exposed to UV radiation for 20, 40, and 60 min, and mutants were subsequently screened for more rapid growth on standard potato dextrose agar. From a screen of >6,000 colonies, mutants were selected based on larger colony diameters as compared to the wild-type parent. Insect bioassays using the diamondback moth, Plutella xylostella, revealed one mutant, designated as MaUV-40.1 as displaying both more rapid growth and increased virulence. The mean lethal time to kill (LT50 using 106 conidia/ml) was 57.6 and 115.4 h for the MaUV-40.1 mutant and wild-type strains, respectively. Total conidial production, UV and thermal tolerances of the MaUV-40.1 strain were increased, but a reduced secretome was seen for the mutant compared to wild type. Analyses of culture supernatants indicated significant shifts in secondary metabolite production in the mutant. The insecticidal activity of EthOAc extracts derived from MaUV-40.1 mutant cell-free culture supernatants were ~20 times more potent that wild-type extracts. These data indicate that mutagenesis coupled to a growth screen can be a simple approach to isolate strains with greater stress resistance and virulence and that cell-free extracts may hold promise as an alternative to the living organism for insect control.

  12. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    Science.gov (United States)

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph). Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Mutants of GABA transaminase (POP2 suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh mutants in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Frank Ludewig

    Full Text Available BACKGROUND: The gamma-aminubutyrate (GABA shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD, the mitochondrial enzymes GABA transaminase (GABA-T; POP2 and succinic semialdehyde dehydrogenase (SSADH. We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA, gamma-hydroxybutyrate (GHB and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

  14. Overproduction of delta-endotoxins by sporeless Bacillus thuringiensis mutants obtained by nitrous acid mutagenesis.

    Science.gov (United States)

    Ben Khedher, Saoussen; Zouari, Nabil; Messaddeq, Nadia; Schultz, Patrick; Jaoua, Samir

    2011-01-01

    Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.

  15. A sorghum (Sorghum bicolor mutant with altered carbon isotope ratio.

    Directory of Open Access Journals (Sweden)

    Govinda Rizal

    Full Text Available Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor mutant with a low δ13C characteristic. A mutant (named Mut33 with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT. The back-cross (BC1F1 progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used

  16. Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

    Science.gov (United States)

    Frandoloso, Rafael; Martínez-Martínez, Sonia; Calmettes, Charles; Fegan, Jamie; Costa, Estela; Curran, Dave; Yu, Rong-Hua; Gutiérrez-Martín, César B; Rodríguez-Ferri, Elías F; Moraes, Trevor F; Schryvers, Anthony B

    2015-03-01

    Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Evaluating Genetic Variability of Sorghum Mutant Lines Tolerant to Acid Soil

    Directory of Open Access Journals (Sweden)

    W. Puspitasari

    2012-08-01

    Full Text Available High rainfall in some parts in Indonesia causes soil become acidic. The main constraint of acid soil is phosphor (P deficiency and aluminum (Al toxicity which decrease plant productivity. To overcome this problem, it is important to develop a crop variety tolerant to such conditions. Sorghum is probably one of the potential crops to meet that objective. Sorghum has been reported to have wide adaptability to various agro-ecology and can be used as food and animal feed. Unfortunately, sorghum is not Indonesian origin so its genetic variability is still low. From previous breeding works with induced mutation, some promising mutant lines have been developed. These mutant lines were included in the experiment carried out in Tenjo with soil condition was classified as acid soil with pH 4.8 and exchangeable-Al content 2.43 me/100 g. The objectives of this experiment were to study the magnitude of genetic variability of agronomy and grain quality characters in sorghum in order to facilitate the breeding improvement of the species. Plant materials used in this study were ten genotypes, including 6 mutant lines and 4 control varieties. The randomized block design with three replications was used in the experiment. The genetic variabilities of agronomic and grain quality characters existed among genotypes, such as plant height, number of leaves, stalk diameter, biomass weight, panicle length, grain yield per plant, 100 seed weight and tannin content in the grain. The broad sense heritabilities of agronomic characters were estimated ranging from medium to high. Grain yield showed significantly positive correlation with agronomic characters observed, but it was negatively correlated with protein content

  18. Evaluating Genetic Variability of Sorghum Mutant Lines Tolerant to Acid Soil

    Directory of Open Access Journals (Sweden)

    W. Puspitasari

    2012-12-01

    Full Text Available High rainfall in some parts in Indonesia causes soil become acidic. The main constraint of acid soil is phosphor (P deficiency and aluminum (Al toxicity which decrease plant productivity. To overcome this problem, it is important to develop a crop variety tolerant to such conditions. Sorghum is probably one of the potential crops to meet that objective. Sorghum has been reported to have wide adaptability to various agro-ecology and can be used as food and animal feed. Unfortunately, sorghum is not Indonesian origin so its genetic variability is still low. From previous breeding works with induced mutation, some promising mutant lines have been developed. These mutant lines were included in the experiment carried out in Tenjo with soil condition was classified as acid soil with pH 4.8 and exchangeable-Al content 2.43 me/100 g. The objectives of this experiment were to study the magnitude of genetic variability of agronomy and grain quality characters in sorghum in order to facilitate the breeding improvement of the species. Plant materials used in this study were ten genotypes, including 6 mutant lines and 4 control varieties. The randomized block design with three replications was used in the experiment. The genetic variabilities of agronomic and grain quality characters existed among genotypes, such as plant height, number of leaves, stalk diameter, biomass weight, panicle length, grain yield per plant, 100 seed weight and tannin content in the grain. The broad sense heritabilities of agronomic characters were estimated ranging from medium to high. Grain yield showed significantly positive correlation with agronomic characters observed, but it was negatively correlated with protein content

  19. The Analysis of Pendolino (peo Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

    Directory of Open Access Journals (Sweden)

    Giovanni Cenci

    2015-06-01

    Full Text Available Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo cause telomeric fusions (TFs. The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

  20. The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

    Science.gov (United States)

    Cenci, Giovanni; Ciapponi, Laura; Marzullo, Marta; Raffa, Grazia D; Morciano, Patrizia; Raimondo, Domenico; Burla, Romina; Saggio, Isabella; Gatti, Maurizio

    2015-06-01

    Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

  1. Phospholamban mutants compete with wild type for SERCA binding in living cells.

    Science.gov (United States)

    Gruber, Simon J; Haydon, Suzanne; Thomas, David D

    2012-04-06

    We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca(2+) cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB(M)) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB(M) in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB(M) and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB(M) for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Vibrissaeless mutant rats with a modular representation of innervated sinus hair follicles in the cerebral cortex.

    Science.gov (United States)

    Kuljis, R O

    1992-01-01

    Specialized areas in the cerebral cortex are essential to mediate the various sensory modalities and are crucial to their recovery in disease. We recently observed that prenatal photoreceptor cues are not indispensable for the development of the elaborate modular organization of the primate primary visual (striate) cortex (Kuljis, R. O. and P. Rakic. 1990. Proc. Natl. Acad. Sci. USA 87: 5303-5306). By contrast, the elegant experiments of Woolsey, Van der Loos, and collaborators (Van der Loos, H., and T. A. Woolsey. 1973. Science 179: 395-398; Van der Loos, H. and J. Dörfl. 1978. Neurosci Lett. 7: 23-30; Woolsey, T. A. 1967. John Hopkins Med. J. 121: 91-112; Woolsey, T. A. and H. Van der Loos. 1970. Brain Res. 17: 205-242) indicate that postnatal vibrissal receptor input is necessary for the development of modular organization in the posteromedial barrel subfield (PMBSF) of the rodent somatosensory cortex. The present report is part of a series of studies designed to address the variables that result in seemingly different results in these two models. Here, I address the role of pre- and postnatal tactile experience in the development of the rat homologue of the mouse PMBSF using mutants that lack vibrissae. Mutants exhibit cytoarchitectonic units in layer IV similar to those in controls, as revealed by NissI stains and histochemistry for succinate dehydrogenase and cytochrome oxidase. Sections from flat mounts of the vibrissal pad reveal that all mutants contain vibrissal follicles with stumps of sinus hairs in a geometric array and number similar to that in controls, and that the follicles are innervated heavily by fascicles of fibers from the infraorbital nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Dominant negative mutant Cyclin T1 proteins inhibit HIV transcription by specifically degrading Tat

    Directory of Open Access Journals (Sweden)

    Okamoto Takashi

    2008-07-01

    Full Text Available Abstract Background The positive transcription elongation factor b (P-TEFb is an essential cellular co-factor for the transcription of the human immunodeficiency virus type 1 (HIV-1. The cyclin T1 (CycT1 subunit of P-TEFb associates with a viral protein, Tat, at the transactivation response element (TAR. This represents a critical and necessary step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies. Results To create effective inhibitors of HIV transcription, mutant CycT1 proteins were constructed based upon sequence similarities between CycT1 and other cyclin molecules, as well as the defined crystal structure of CycT1. One of these mutants, termed CycT1-U7, showed a potent dominant negative effect on Tat-dependent HIV transcription despite a remarkably low steady-state expression level. Surprisingly, the expression levels of Tat proteins co-expressed with CycT1-U7 were significantly lower than Tat co-expressed with wild type CycT1. However, the expression levels of CycT1-U7 and Tat were restored by treatment with proteasome inhibitors. Concomitantly, the dominant negative effect of CycT1-U7 was abolished by these inhibitors. Conclusion These results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription that could potentially be used in the design of anti-viral therapy.

  4. Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity

    Directory of Open Access Journals (Sweden)

    Vijayata Singh

    2017-08-01

    Full Text Available The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1 functions as a co-receptor with several receptor kinases including the brassinosteroid (BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1, which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2 and EF-TU RECEPTOR (EFR, respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F in the bak1-4 null mutant. Neither BAK1 (Y610F transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive

  5. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila.

    Science.gov (United States)

    Seki, Yuuichi; Tanimura, Teiichi

    2014-09-01

    A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf 01) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.

  6. Dynamic void distribution in myoglobin and five mutants

    Science.gov (United States)

    Jiang, Yingying; Kirmizialtin, Serdal; Sanchez, Isaac C.

    2014-02-01

    Globular proteins contain cavities/voids that play specific roles in controlling protein function. Elongated cavities provide migration channels for the transport of ions and small molecules to the active center of a protein or enzyme. Using Monte Carlo and Molecular Dynamics on fully atomistic protein/water models, a new computational methodology is introduced that takes into account the protein's dynamic structure and maps all the cavities in and on the surface. To demonstrate its utility, the methodology is applied to study cavity structure in myoglobin and five of its mutants. Computed cavity and channel size distributions reveal significant differences relative to the wild type myoglobin. Computer visualization of the channels leading to the heme center indicates restricted ligand access for the mutants consistent with the existing interpretations. The new methodology provides a quantitative measure of cavity structure and distributions and can become a valuable tool for the structural characterization of proteins.

  7. Characterizing visible and invisible cell wall mutant phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Carpita, Nicholas C.; McCann, Maureen C.

    2015-04-06

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

  8. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...... in which apoptosis can be studied using the novel, temperature sensitive mutant, cdc77. The cdc77 cells are defective in a G1 process, and die show the characteristc signs of apoptosis: condensation of the chromatin, degradation of the inner nuclear membrane, dilation of the space between the nuclear...

  9. Multi-level evaluation of Escherichia coli polyphosphate related mutants using global transcriptomic, proteomic and phenomic analyses.

    Science.gov (United States)

    Varas, Macarena; Valdivieso, Camilo; Mauriaca, Cecilia; Ortíz-Severín, Javiera; Paradela, Alberto; Poblete-Castro, Ignacio; Cabrera, Ricardo; Chávez, Francisco P

    2017-04-01

    Polyphosphate (polyP) is a linear biopolymer found in all living cells. In bacteria, mutants lacking polyphosphate kinase 1 (PPK1), the enzyme responsible for synthesis of most polyP, have many structural and functional defects. However, little is known about the causes of these pleiotropic alterations. The link between ppk1 deletion and those numerous phenotypes observed can be the result of complex molecular interactions that can be elucidated via a systems biology approach. By integrating different omics levels (transcriptome, proteome and phenome), we described the functioning of various metabolic pathways among Escherichia coli polyphosphate mutant strains (Δppk1, Δppx, and ΔpolyP). Bioinformatic analyses reveal the complex metabolic and regulatory bases of the phenotypes unique to polyP mutants. Our results suggest that during polyP deficiency (Δppk1 mutant), metabolic pathways needed for energy supply are up-regulated, including fermentation, aerobic and anaerobic respiration. Transcriptomic and q-proteomic contrasting changes between Δppk1 and Δppx mutant strains were observed in those central metabolic pathways and confirmed by using Phenotypic microarrays. In addition, our results suggest a regulatory connection between polyP, second messenger metabolism, alternative Sigma/Anti-Sigma factors and type-II toxin-antitoxin (TA) systems. We suggest a broader role for polyP via regulation of ATP-dependent proteolysis of type II toxin-antitoxin system and alternative Sigma/Anti-Sigma factors, that could explain the multiple structural and functional deficiencies described due to alteration of polyP metabolism. Understanding the interplay of polyP in bacterial metabolism using a systems biology approach can help to improve design of novel antimicrobials toward pathogens. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Development and evaluation of drought resistant mutant germ ...

    African Journals Online (AJOL)

    The aim of this project was to select cowpea plants with improved levels of drought resistance without alteration to the colour of the testa or the growth form. Seed from M2 to M5 generations (M = mutant) were used in the study. The M2 to M4 seeds were planted and evaluated in wooden boxes in the greenhouse and in the ...

  11. Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes.

    Directory of Open Access Journals (Sweden)

    Mohammad Haeri

    Full Text Available Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS, accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. Rho(P23H-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H, suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.

  12. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine

  13. Lactate dehydrogenase A silencing in IDH mutant gliomas.

    Science.gov (United States)

    Chesnelong, Charles; Chaumeil, Myriam M; Blough, Michael D; Al-Najjar, Mohammad; Stechishin, Owen D; Chan, Jennifer A; Pieper, Russell O; Ronen, Sabrina M; Weiss, Samuel; Luchman, H Artee; Cairncross, J Gregory

    2014-05-01

    Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant ((mt)) and IDH wild-type ((wt)) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH(mt) gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH(mt) derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH(wt)), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH(mt) glioblastomas. To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH(mt) gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.

  14. Wheat ABA-insensitive mutants result in reduced grain dormancy

    Science.gov (United States)

    Schramm, Elizabeth C.; Nelson, Sven K.

    2014-01-01

    This paper describes the isolation of wheat mutants in the hard red spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic acid (ABA) during seed germination. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature seeds. Wheat sensitivity to ABA gradually decreases with dry after-ripening. Scarlet grain normally fails to germinate when fully dormant, shows ABA sensitive germination when partially after-ripened, and becomes ABA insensitive when after-ripened for 8–12 months. Scarlet ABA-insensitive (ScABI) mutants were isolated based on the ability to germinate on 5 µM ABA after only 3 weeks of after-ripening, a condition under which Scarlet would fail to germinate. Six independent seed-specific mutants were recovered. ScABI 1, ScABI2, ScABI3 and ScABI4 are able to germinate more efficiently than Scarlet at up to 25 µM ABA. The two strongest ABA insensitive lines, ScABI3 and ScABI4, both proved to be partly dominant suggesting that they result from gain-of-function mutations. The ScABI1, ScABI2, ScABI3, ScABI4, and ScABI5 mutants after-ripen more rapidly than Scarlet. Thus, ABA insensi-tivity is associated with decreased grain dormancy in Scarlet wheat. This suggests that ABA sensitivity is an important factor controlling grain dormancy in wheat, a trait that impacts seedling emergence and pre-harvest sprouting resistance. PMID:25431501

  15. Xylitol production by a Pichia stipitis D-xylulokinase mutant

    Science.gov (United States)

    Yong-Su Jin; Jose Cruz; Thomas W. Jeffries

    2005-01-01

    Xylitol production by Pichia stipitis FPL-YS30, a xyl3-Ä1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-Ä1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g...

  16. Temperature Sensitivity of Neural Tube Defects in Zoep Mutants.

    Science.gov (United States)

    Ma, Phyo; Swartz, Morgan R; Kindt, Lexy M; Kangas, Ashley M; Liang, Jennifer Ostrom

    2015-12-01

    Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation.

  17. Genes and Alcohol Consumption: Studies with Mutant Mice

    Science.gov (United States)

    Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

    2017-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

  18. Drosophila melanogaster White Mutant w1118 Undergo Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    María José Ferreiro

    2018-01-01

    Full Text Available Key scientific discoveries have resulted from genetic studies of Drosophila melanogaster, using a multitude of transgenic fly strains, the majority of which are constructed in a genetic background containing mutations in the white gene. Here we report that white mutant flies from w1118 strain undergo retinal degeneration. We observed also that w1118 mutants have progressive loss of climbing ability, shortened life span, as well as impaired resistance to various forms of stress. Retinal degeneration was abolished by transgenic expression of mini-white+ in the white null background w1118. We conclude that beyond the classical eye-color phenotype, mutations in Drosophila white gene could impair several biological functions affecting parameters like mobility, life span and stress tolerance. Consequently, we suggest caution and attentiveness during the interpretation of old experiments employing white mutant flies and when planning new ones, especially within the research field of neurodegeneration and neuroprotection. We also encourage that the use of w1118 strain as a wild-type control should be avoided.

  19. Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Radwanski, E R; Barczak, A J; Last, R L

    1996-12-13

    Three mutations in the Arabidopsis thaliana gene encoding the alpha subunit of tryptophan synthase were isolated by selection for resistance to 5-methylanthranilate or 5-fluoroindole, toxic analogs of tryptophan pathway intermediates. Plants homozygous for trp3-1 and trp3-2 are light-conditional tryptophan auxotrophs, while trp3-100 is a more leaky mutant. Genetic complementation crosses demonstrated that the three mutations are allelic to each other, and define a new complementation group. All three mutants have decreased steady-state levels of tryptophan synthase alpha protein, and the trp3-100 polypeptide exhibits altered electrophoretic mobility. All three mutations were shown to be in the TSA1 (tryptophan synthase alpha subunit) structural gene by several criteria. Firstly, the trp3-1 mutation is linked to TSA1 on the bottom of chromosome 3. Secondly, the trp3-1 mutation was complemented when transformed with the wild-type TSA1 gene. Finally, DNA sequence analysis of the TSA1 gene revealed a single transition mutation in each trp3 mutant.

  20. Coloboma hyperactive mutant exhibits delayed neurobehavioral developmental milestones.

    Science.gov (United States)

    Heyser, C J; Wilson, M C; Gold, L H

    1995-11-21

    The coloboma mutation (Cm) is a neutron-irradiation induced gene deletion located on the distal portion of mouse chromosome 2. This deletion region includes a gene encoding the synaptic vesicle docking fusion protein, synaptosomal-associated protein of 25 kDa (SNAP-25). The resulting mutation is semi-dominant with heterozygote mice exhibiting a triad of phenotypic abnormalities that comprise profound spontaneous hyperactivity, head bobbing and a prominent eye dysmorphology. Because the expression pattern of two SNAP-25 isoforms begins to change during the first postnatal week, neurobehavioral developmental milestones were examined in order to determine if the expression of the coloboma behavioral phenotype could be detected during this period of postnatal development. The early classification of coloboma mutant offspring may help to further describe the penetrance of this mutation as well as the contribution of developmental changes to the adult behavioral phenotype. The coloboma mutation resulted in delays in some tests of complex motor skills including righting reflex and bar holding. In addition, coloboma mutants were characterized by body weight differences (first appearance day 7) and hyperreactivity to touch (day 11) and head bobbing (day 14). These data demonstrate disruptions in the time course of attaining developmental milestones in coloboma mutants and provide further evidence supporting the hypotheses that alterations in Snap gene expression are associated with functional behavioral consequences in developing offspring.

  1. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

    Directory of Open Access Journals (Sweden)

    Pavithra eViswanath

    2016-03-01

    Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  2. Molecular Imaging of Metabolic Reprograming in Mutant IDH Cells.

    Science.gov (United States)

    Viswanath, Pavithra; Chaumeil, Myriam M; Ronen, Sabrina M

    2016-01-01

    Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an "oncometabolite," inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  3. Functional Analysis of Jasmonates in Rice through Mutant Approaches

    Directory of Open Access Journals (Sweden)

    Rohit Dhakarey

    2016-03-01

    Full Text Available Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes participating in its biosynthesis were characterized. In the past 15 years, mutants and transgenic plants affected in the jasmonate pathway became available in rice and facilitate studies on the functions of this hormone in an important crop. Those functions are partially conserved compared to other plant species, and include roles in fertility, response to mechanical wounding and defense against herbivores. However, new and surprising functions have also been uncovered by mutant approaches, such as a close link between light perception and the jasmonate pathway. This was not only useful to show a phenomenon that is unique to rice but also helped to establish this role in plant species where such links are less obvious. This review aims to provide an overview of currently available rice mutants and transgenic plants in the jasmonate pathway and highlights some selected roles of jasmonate in this species, such as photomorphogenesis, and abiotic and biotic stress.

  4. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    Science.gov (United States)

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Genes and Alcohol Consumption: Studies with Mutant Mice.

    Science.gov (United States)

    Mayfield, J; Arends, M A; Harris, R A; Blednov, Y A

    2016-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. © 2016 Elsevier Inc. All rights reserved.

  6. Virulence of Burkholderia mallei quorum-sensing mutants.

    Science.gov (United States)

    Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard; Greenberg, E Peter

    2013-05-01

    Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved.

  7. Histological Characterization of the Dicer1 Mutant Zebrafish Retina

    Directory of Open Access Journals (Sweden)

    Saeed Akhtar

    2015-01-01

    Full Text Available DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA and RNA-interference (RNAi functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE cells are normal with no sign of degeneration at the age of 20 months.

  8. Recombination Phenotypes of Escherichia coli greA Mutants

    Directory of Open Access Journals (Sweden)

    Poteete Anthony R

    2011-03-01

    Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

  9. Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

    Science.gov (United States)

    Gardiol, A; Arias, A; Cerveñansky, C; Gaggero, C; Martínez-Drets, G

    1980-10-01

    A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.

  10. Pattern formation mechanisms in motility mutants of Myxococcus xanthus

    CERN Document Server

    Starruss, Joern; Jakovljevic, Vladimir; Sogaard-Andersen, Lotte; Deutsch, Andreas; Baer, Markus

    2016-01-01

    Formation of spatial patterns of cells is a recurring theme in biology and often depends on regulated cell motility. Motility of M. xanthus depends on two motility machineries: the S-engine and A-engine. Moving M. xanthus cells can organize into spreading colonies or spore-filled fruiting bodies depending on their nutritional status. To understand these two pattern formation processes and the contributions by the two motility machineries, as well as cell reversal, we analyze spatial self-organization in 3 strains: i) a mutant that moves unidirectionally without reversing by the A-motility system only, ii) a unidirectional mutant that is also equipped with the S-motility system, and iii) the wild-type that, in addition to the two motility systems, reverses its direction of movement. The mutant moving by the A-engine illustrates that collective motion in the form of large moving clusters can arise in gliding bacteria due to steric interactions of the rod-shaped cells, without the need of invoking any biochemica...

  11. Inhibition of Rhizobium etli Polysaccharide Mutants by Phaseolus vulgaris Root Compounds

    OpenAIRE

    Eisenschenk, Linda; Diebold, Ronald; Perez-Lesher, Jeanett; Peterson, Andrew C.; Kent Peters, N.; Noel, K. Dale

    1994-01-01

    Crude bean root extracts of Phaseolus vulgaris were tested for inhibition of the growth of several polysaccharide mutants of Rhizobium etli biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher l...

  12. Bacterial adherence to eucaryotic cells: isolation of lymphocyte-binding mutants.

    Science.gov (United States)

    Mayer, E P; Teodorescu, M

    1980-01-01

    A procedure for obtaining bacterial mutants that bind to eucaryotic cells is described. This procedure takes advantage of the ability of the mutants to obtain a required nutrient from the eucaryotic cells. We used this procedure to isolate mutants of Escherichia coli that bind to mouse lymphocytes. We show that the mutants identify some immunoglobulin-bearing lymphocytes and some non-immunoglobulin-bearing lymphocytes. PMID:6995342

  13. Bacterial adherence to eucaryotic cells: isolation of lymphocyte-binding mutants.

    OpenAIRE

    Mayer, E P; Teodorescu, M

    1980-01-01

    A procedure for obtaining bacterial mutants that bind to eucaryotic cells is described. This procedure takes advantage of the ability of the mutants to obtain a required nutrient from the eucaryotic cells. We used this procedure to isolate mutants of Escherichia coli that bind to mouse lymphocytes. We show that the mutants identify some immunoglobulin-bearing lymphocytes and some non-immunoglobulin-bearing lymphocytes.

  14. Selection of 5-fluorocytosine-resistant mutants from an Aspergillus niger citric acid-producing strain

    Directory of Open Access Journals (Sweden)

    Conte Ana Paula de F.

    2003-01-01

    Full Text Available Mutants of Aspergillus niger N402, induced by UV mutagenesis, were selected and tested for resistance or sensitivity to 5-fluorocytosine. Some mutants showed increased citric acid production, which did not correlate with the intracellular amount of protein or ammonium ion. The resistance to 5-fluorocytosine proved to be a rational approach for isolation of new mutants with improved production of citric acid. The best mutant (FR13 accumulated double the amount of citric acid produced by the parental strain.

  15. Energetic basis for drug resistance of HIV-1 protease mutants against amprenavir

    Science.gov (United States)

    Kar, Parimal; Knecht, Volker

    2012-02-01

    Amprenavir (APV) is a high affinity (0.15 nM) HIV-1 protease (PR) inhibitor. However, the affinities of the drug resistant protease variants V32I, I50V, I54V, I54M, I84V and L90M to amprenavir are decreased 3 to 30-fold compared to the wild-type. In this work, the popular molecular mechanics Poisson-Boltzmann surface area method has been used to investigate the effectiveness of amprenavir against the wild-type and these mutated protease variants. Our results reveal that the protonation state of Asp25/Asp25' strongly affects the dynamics, the overall affinity and the interactions of the inhibitor with individual residues. We emphasize that, in contrast to what is often assumed, the protonation state may not be inferred from the affinities but requires pKa calculations. At neutral pH, Asp25 and Asp25' are ionized or protonated, respectively, as suggested from pKa calculations. This protonation state was thus mainly considered in our study. Mutation induced changes in binding affinities are in agreement with the experimental findings. The decomposition of the binding free energy reveals the mechanisms underlying binding and drug resistance. Drug resistance arises from an increase in the energetic contribution from the van der Waals interactions between APV and PR (V32I, I50V, and I84V mutant) or a rise in the energetic contribution from the electrostatic interactions between the inhibitor and its target (I54M and I54V mutant). For the V32I mutant, also an increased free energy for the polar solvation contributes to the drug resistance. For the L90M mutant, a rise in the van der Waals energy for APV-PR interactions is compensated by a decrease in the polar solvation free energy such that the net binding affinity remains unchanged. Detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.

  16. Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7

    DEFF Research Database (Denmark)

    Czene, Aniko; Toth, Eszter; Gyurcsik, Bela

    2013-01-01

    the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C* of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method...

  17. A Molecular Dynamics Investigation into the Mechanisms of Alectinib Resistance of Three ALK Mutants.

    Science.gov (United States)

    He, Muyang; Li, Weikang; Zheng, Qingchuan; Zhang, Hongxing

    2018-01-11

    Alectinib, a highly selective next-genetation anaplastic lymphoma kinase (ALK) inhibitor, has demonstrated promising antitumor activity in patients with ALK-positive non-small cell lung carcinomas (NSCLC). However, the therapeutic benefits of alectinib is inescapably hampered by the development of acquired resistant mutations in ALK. Despite the availability of ample experimental mutagenesis data, the molecular origin and the structural motifs under alectinib binding affinity deficiencies are still ambiguous. Here, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) calculation approaches were employed to elucidate the mechanisms of alectinib resistance induced by the mutations I1171N, V1180L, and L1198F. The MD results reveal that the studied mutations could trigger the dislocation of alectinib as well as conformational changes at the inhibitor binding site, thus induce the interactional changes between alectinib and mutants. The most influenced regions are the ligand binding entrance and the hinge region, which are considered to be the dominant binding motifs accounting for the binding affinity loss in mutants. The "key and lock mechanism" between the ethyl group at position 9 of alectinib and a recognition cavity in the hinge region of ALK is presented to illustrate the major molecular origin of drug resistance. Our results provide mechanistic insight into the effect of ALK mutations resistant to alectinib, which could contribute to further rational design of inhibitors to combat the acquired resistance. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant.

    Science.gov (United States)

    Cutzu, Raffaela; Coi, Annalisa; Rosso, Fulvia; Bardi, Laura; Ciani, Maurizio; Budroni, Marilena; Zara, Giacomo; Zara, Severino; Mannazzu, Ilaria

    2013-06-01

    In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2) = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l(-1)) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.

  19. Pleurotus sajor-caju HSP100 complements a thermotolerance defect in hsp104 mutant Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Jin-Ohk; Jeong, Mi-Jeong; Kwon, Tack-Ryun; Lee, Seung-Kon; Byun, Myung-Ok; Chung, Ill-Min; Park, Soo-Chul

    2006-06-01

    A putative Hsp100 gene was cloned from the fungus Pleurotus sajor-caju. mRNA expression studies demonstrated that this gene (designated PsHsp100) is highly induced by high temperature,induced less strongly by exposure to ethanol, and not induced by drought or salinity. Heat shock induction is detectable at 37 degrees C and reaches a maximum level at 42 degrees C. PsHsp100 mRNA levels sharply increased within 15 min of exposure to high temperature, and reached a maximum expression level at 2 h that was maintained for several hours. These results indicate that PsHsp100 could work at an early step in thermotolerance. To examine its function, PsHsp100 was transformed into a temperature-sensitive hsp104 deletion mutant Saccharomycetes cerivisiae strain to test the hypothesis that PsHSP100 is an protein that functions in thermotolerance. Overexpression of PsHSP100 complemented the thermotolerance defect of the hsp104 mutant yeast, allowing them being survive even at 50 degree C for 4 h. These results indicate that PsHSP100 protein is functional as an HSP100 in yeast and could play and important role in thermotolerance in P. sajor-caju.

  20. Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08*

    Science.gov (United States)

    Zhang, Xiao-yan; Peng, Yong; Su, Zhong-rui; Chen, Qi-he; Ruan, Hui; He, Guo-qing

    2013-01-01

    Biotransformation of phytosterol (PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper. The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology. Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M. neoaurum ZJUVN-08 strain. Results showed that molar ratio of hydroxypropyl-β-cyclodextrin (HP-β-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production. By analyzing the statistical model of three-dimensional surface plot, the optimal process conditions were observed at 0.1 g/L inducer, pH 7.0, molar ratio of HP-β-CD to PS 1.92:1, 8.98 g/L PS, and at 120 h of incubation time. Under these conditions, the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized (5.99 g/L), while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized (84.03%). The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results. It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process. PMID:23365012

  1. Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08.

    Science.gov (United States)

    Zhang, Xiao-yan; Peng, Yong; Su, Zhong-rui; Chen, Qi-he; Ruan, Hui; He, Guo-qing

    2013-02-01

    Biotransformation of phytosterol (PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper. The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology. Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M. neoaurum ZJUVN-08 strain. Results showed that molar ratio of hydroxypropyl-β-cyclodextrin (HP-β-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production. By analyzing the statistical model of three-dimensional surface plot, the optimal process conditions were observed at 0.1 g/L inducer, pH 7.0, molar ratio of HP-β-CD to PS 1.92:1, 8.98 g/L PS, and at 120 h of incubation time. Under these conditions, the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized (5.99 g/L), while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized (84.03%). The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results. It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process.

  2. Evidence for dynamic network regulation of Drosophila photoreceptor function from mutants lacking the neurotransmitter histamine

    Directory of Open Access Journals (Sweden)

    An eDau

    2016-03-01

    Full Text Available Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdcJK910 mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdcJK910 photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdcJK910 photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdcJK910 R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdcJK910 mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.

  3. Gγ1 + Gγ2 ≠ Gβ: Heterotrimeric G Protein Gγ-Deficient Mutants Do Not Recapitulate All Phenotypes of Gβ-Deficient Mutants1[C][W][OA

    Science.gov (United States)

    Trusov, Yuri; Zhang, Wei; Assmann, Sarah M.; Botella, José Ramón

    2008-01-01

    Heterotrimeric G proteins are signaling molecules ubiquitous among all eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains one Gα (GPA1), one Gβ (AGB1), and two Gγ subunit (AGG1 and AGG2) genes. The Gβ requirement of a functional Gγ subunit for active signaling predicts that a mutant lacking both AGG1 and AGG2 proteins should phenotypically resemble mutants lacking AGB1 in all respects. We previously reported that Gβ- and Gγ-deficient mutants coincide during plant pathogen interaction, lateral root development, gravitropic response, and some aspects of seed germination. Here, we report a number of phenotypic discrepancies between Gβ- and Gγ-deficient mutants, including the double mutant lacking both Gγ subunits. While Gβ-deficient mutants are hypersensitive to abscisic acid inhibition of seed germination and are hyposensitive to abscisic acid inhibition of stomatal opening and guard cell inward K+ currents, none of the available Gγ-deficient mutants shows any deviation from the wild type in these responses, nor do they show the hypocotyl elongation and hook development defects that are characteristic of Gβ-deficient mutants. In addition, striking discrepancies were observed in the aerial organs of Gβ- versus Gγ-deficient mutants. In fact, none of the distinctive traits observed in Gβ-deficient mutants (such as reduced size of cotyledons, leaves, flowers, and siliques) is present in any of the Gγ single and double mutants. Despite the considerable amount of phenotypic overlap between Gβ- and Gγ-deficient mutants, confirming the tight relationship between Gβ and Gγ subunits in plants, considering the significant differences reported here, we hypothesize the existence of new and as yet unknown elements in the heterotrimeric G protein signaling complex. PMID:18441222

  4. Inverse polymerase chain reaction for rapid gene isolation in Arabidopsis thaliana insertion mutants

    NARCIS (Netherlands)

    Vanderhaeghen, R.; Scheres, B.J.G.; Montagu, M. van; Lijsebetten, M. van

    1992-01-01

    Recently, many mutants have been isolated in the model plant Arabidopsis thaliana by the insertion of the Agrobacterium tumefaciens T-DNA into the plant genome. Instead of applying Southern analysis on these insertion mutants and to avoid the construction of mutant- derived genomic libraries,

  5. Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli

    DEFF Research Database (Denmark)

    Brown, S; Brickman, E R; Beckwith, J

    1981-01-01

    We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon inser...

  6. Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

    NARCIS (Netherlands)

    van Vugt-Lussenburg, B.M.A.; Damsten, M.C.; Maasdijk, D.M.; Vermeulen, N.P.E.; Commandeur, J.N.M.

    2006-01-01

    Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine,

  7. Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion

    Science.gov (United States)

    James M. Slavicek; Melissa J. Mercer; Dana Pohlman; Mary Ellen Kelly; David S. Bischoff

    1998-01-01

    In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. Ld

  8. Differential analysis in Proteome of Space Induced Rice and Soybean Mutants

    Science.gov (United States)

    Wang, W.; Lu, B.; Gu, D.; Han, S.; Gao, Y.; Sun, Y.

    To investigate the change trends of proteome induced in space environment we chose 3 Rice mutants 2 Soybean mutants and the seeds which were selected as high yields high tillering rice blast resistance soybean insect pest resistance and wider leaf shape individually after abroad Recoverable Satellite JB-1 for 15 days in 1996 and their corresponding controls Two-dimensional gel electrophoresis 2-D with Coomassie Brilliant Blue staining and PDQuest TM software analysis found that In 6 rice samples 329 pm 35 protein spots were detected in controls whereas 298 pm 37 protein spots detected in mutants representing a 9 decrease 69 pm 27 protein spots were lost in mutants while 37 pm 14 protein spots appeared additionally showing 11 protein spots were lost in mutants 58 protein spots were significantly regulated in mutants with 16 pm 7 up- and 42 pm 18 down-regulated which occupied 5 and 14 of the total average mutants spots separately In 3 soybean leaf samples 263 pm 12 protein spots were detected in controls whereas 255 pm 20 protein spots detected in mutants representing a 3 decrease 49 pm 10 protein spots were lost in mutants while 36 pm 16 protein spots appeared additionally showing 5 protein spots lost in mutants 51 protein spots were significantly regulated in mutants with 25 pm 7 up- and 26 pm 15 down-regulated which occupied 9 8 and 10 2 of the total average mutants spots separately In 3 soybean seed samples 208 pm 41 protein spots were

  9. Isolation, characterization, and expression analyses of tryptophan aminotransferase genes in a maize dek18 mutant

    Science.gov (United States)

    The dek18 mutant of maize has decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology in maize seed development. Seeds of the dek18 mutants are smaller compared to wild type seeds and the vegetative d...

  10. Grain product of 34 soya mutant lines;Rendimiento de grano de 34 lineas mutantes de soya

    Energy Technology Data Exchange (ETDEWEB)

    Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E. [Colegio Superior Agropecuario del Estado de Guerrero, Vicente Guerrero No. 81, Col. Centro, 40000 Iguala, Guerrero (Mexico); Cervantes S, T. [Instituto de Recursos Geneticos y Productividad, Colegio de Posgraduados, Carretera Mexico-Texcoco Km. 36.5, Montecillo, 56230 Texcoco, Estado de Mexico (Mexico); De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A. [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2009-07-01

    This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R{sub 4}M{sub 18}) for human consumption and this way to select the 2 better lines. The genetic materials were obtained starting from the variety ISAAEG-B M2 by means of the application of recurrent radiation with Co{sup 60} gammas, to a dose of 350 Gray for the first two generations and both later to 200 Gray and selection during 17 cycles, being obtained the 34 better lines mutants with agronomic characteristic wanted and good flavor. The obtained results were that the mutant lines L{sub 25} and L{sub 32} produced the major quantity in branches/plant number with 7.5 and 7.25, pods/plant number with 171.25 and 167, grains/plant number with 350.89 and 333.07 and grain product (ton/ha) to 15% of humidity 5.15 and 4.68 ton/ha, respectively. (Author)

  11. The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant.

    Science.gov (United States)

    Tiruneh, Bayu Sisay; Kim, Byung-Hoon; Gallie, Daniel R; Roy, Bijoyita; von Arnim, Albrecht G

    2013-12-30

    Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. These data represent the first genome

  12. Functional studies of elongation factor Tu from Escherichia coli : Site-directed mutagenesis and antibiotic action

    NARCIS (Netherlands)

    Krab, Ivo Maarten

    2001-01-01

    This PhD thesis describes several studies into the structure and function of Escherichia coli Elongation Factor Tu (EF-Tu). EF-Tu plays a central role in the bacterial protein synthesis machinery as the carrier of "coded building blocks" for protein synthesis, aminoacylated tRNA (aa-tRNA). Without

  13. Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

    DEFF Research Database (Denmark)

    Liu, Wei; Kavaliauskas, Darius; Schrader, Jared

    2014-01-01

    ) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput...

  14. Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

    Science.gov (United States)

    Rao, T R; Slobin, L I

    1987-02-01

    When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.

  15. Functional consequences of T-stem mutations in E. coli tRNA Thr UGU in vitro and in vivo

    DEFF Research Database (Denmark)

    Saks, Margaret E; Sanderson, Lee E; Choi, Daniel S

    2011-01-01

    The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNAThrUGU were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound...

  16. One-step purification of E. coli elongation factor Tu

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.; Degn, B

    1993-01-01

    The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one...

  17. Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

    Science.gov (United States)

    Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

    2017-03-01

    In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. The eta7/csn3-3 auxin response mutant of Arabidopsis defines a novel function for the CSN3 subunit of the COP9 signalosome.

    Directory of Open Access Journals (Sweden)

    He Huang

    Full Text Available The COP9 signalosome (CSN is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF(TIR1/AFB ubiquitin-ligase (deneddylation. Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3, designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF(TIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.

  19. Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

    Science.gov (United States)

    Silva, Jerson L; Cino, Elio A; Soares, Iaci N; Ferreira, Vitor F; A P de Oliveira, Guilherme

    2018-01-16

    ) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation.

  20. Leaf Rolling and Stem Fasciation in Grass Pea (Lathyrus sativus L.) Mutant Are Mediated through Glutathione-Dependent Cellular and Metabolic Changes and Associated with a Metabolic Diversion through Cysteine during Phenotypic Reversal

    OpenAIRE

    Talukdar, Dibyendu; Talukdar, Tulika

    2014-01-01

    A Lathyrus sativus L. mutant isolated in ethylmethane sulfonate-treated M2 progeny of mother variety BioL-212 and designated as rlfL-1 was characterized by inwardly rolled-leaf and stem and bud fasciations. The mutant exhibited karyomorphological peculiarities in both mitosis and meiosis with origin of aneuploidy. The mitosis was vigorous with high frequency of divisional cells and their quick turnover presumably steered cell proliferations. Significant transcriptional upregulations of cyste...

  1. New mutants and their chromosome assignment in the fly Megaselia scalaris.

    Science.gov (United States)

    Traut, W; Traut, G; Mertl, H G; Egelhaaf, A

    1994-01-01

    We describe six new mutants, spontaneous or EMS-induced, and a previously isolated radiation-induced mutant of Megaselia scalaris Loew (Diptera, Phoridae). Five are eye-color mutants, one affects the segmentation of the abdomen, and one disturbs the regular form and arrangement of ommatidia in the compound eye. A complementation test exposed two pairs of alleles among the five eye-color mutants, leaving three different loci that affect eye pigmentation. Two of them influence the amount of xanthommatin synthesized, and one blocks the ommochrome pathway at the kynurenine step. We established chromosome assignment of the mutants by crossbreeding them with Y chromosome-autosome translocation strains.

  2. Xanthine Dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity.

    Science.gov (United States)

    Browder, L W; Tucker, L; Wilkes, J

    1982-02-01

    Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin and cin mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.

  3. Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants.

    Directory of Open Access Journals (Sweden)

    Yuanli Liu

    Full Text Available Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS characteristic of treatment with quinolone (oxolinic acid. These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability.

  4. Analysis of Escherichia coli mutants with a linear respiratory chain.

    Directory of Open Access Journals (Sweden)

    Sonja Steinsiek

    Full Text Available The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important.

  5. The antiandrogenic effect of finasteride against a mutant androgen receptor

    Science.gov (United States)

    Chhipa, Rishi Raj; Zhang, Haitao; Ip, Clement

    2011-01-01

    Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild-type AR. When PC-3 cells, which are AR-null, were transfected with either the wild-type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy. PMID:21386657

  6. A sialidase mutant displaying trans-sialidase activity.

    Science.gov (United States)

    Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M; Frasch, Alberto Carlos C

    2005-01-28

    Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.

  7. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  8. Mutant prevention concentrations of daptomycin for Enterococcus faecium clinical isolates.

    Science.gov (United States)

    Sinel, Clara; Jaussaud, Clara; Auzou, Michel; Giard, Jean-Christophe; Cattoir, Vincent

    2016-10-01

    Owing to the emergence of vancomycin-resistant Enterococcus faecium, treatment of enterococcal infections has become challenging. Although spontaneous in vitro resistance frequencies are low, the emergence of resistance is increasingly reported during daptomycin therapy. The mutant selection window (MSW), comprised between the minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC), corresponds to the concentration range within which resistant mutants may be selected. Since no data are available for enterococci, the aim of this study was to determine MPCs and MSWs for 12 representative E. faecium clinical isolates. MICs and MPCs were determined by broth microdilution and agar dilution methods, respectively. A basic MSW-derived pharmacodynamic analysis was also performed using mean maximum plasma concentration (Cmax) values obtained with dosages from 4 to 12 mg/kg. MICs and MPCs of daptomycin ranged from 0.5 to 4 mg/L and from 2 to 32 mg/L, respectively, with no correlation between them. The wideness of MSWs ranged from 2× to 32× MIC. Mean plasma Cmax values of daptomycin were calculated from 55 to 174.5 mg/L when using a dosage from 4 to 12 mg/kg. All Cmax values were above the MPCs whatever the dosage. Taking into account the protein binding of daptomycin (ca. 90%), the unbound fraction Cmax was just within the MSW in 67-92% of strains at recommended dosages (4-6 mg/kg) and was above the MPC for the majority of strains only with the highest dosage (12 mg/kg). This study shows that free daptomycin Cmax values usually fell into MSWs when using lower dosages (<10 mg/kg). Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  9. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Science.gov (United States)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

    2009-01-01

    SUMMARY Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABLT315I mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and pre-clinical evaluation of AP24534, a potent, orally available multi-targeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABLT315I-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML. PMID:19878872

  10. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Energy Technology Data Exchange (ETDEWEB)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  11. clustering common bean mutants based on heterotic groupings ...

    African Journals Online (AJOL)

    ACSS

    diversité génétique entre les haricots mutants, ces groupes (A, B et C) peuvent être considérés comme des groupements hétérotiques. Selon le trait phénotypique considéré, le croisement de deux des génotypes appartenant à des groupes différents peut générer de la vigueur hybride. Par ailleurs, pour créer une variabilité ...

  12. Using PATIMDB to create bacterial transposon insertion mutant libraries.

    Science.gov (United States)

    Urbach, Jonathan M; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M

    2009-04-01

    PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software.

  13. Environmental features determining successful rearing in the mutant mouse staggerer.

    Science.gov (United States)

    Guastavino, J M

    1984-02-01

    The mutant mouse staggerer is unable to rear her pups to weaning unless special precautions are taken. The following environmental conditions were found to contribute to compensate for the maternal behavioral deficits of the staggerer: (1) the foster pups used in a constraining box to stimulate the lactating staggerer mother are 4 days old. (2) The mother is enforced to stay in close physical contact with these pups for at least 12 hours immediately after delivery. (3) The staggerer pups are transferred to a normal lactating mother to suckle her for the first 12 hours of life.

  14. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...

  15. Relationships between PSII-independent hydrogen bioproduction and starch metabolism as evidenced from isolation of starch catabolism mutants in the green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Chochois, Vincent; Constans, Laure; Beyly, Audrey; Soliveres, Melanie; Peltier, Gilles; Cournac, Laurent [CEA, DSV, IBEB, Laboratoire de Bioenergetique et Biotechnologie des Bacteries and Microalgues, Saint Paul Lez Durance, F-13108 (France); CNRS, UMR Biologie Vegetale and Microbiologie Environnementales, Saint Paul lez Durance, F-13108 (France); Aix-Marseille Universite, Saint Paul lez Durance, F-13108 (France); Dauvillee, David; Ball, Steven [Univ Lille Nord de France, F-59000 Lille (France); USTL, UGSF, F-59650 Villeneuve d' Ascq (France); CNRS, UMR 8576, F-59650 Villeneuve d' Ascq (France)

    2010-10-15

    Sulfur deprivation, which is considered as an efficient way to trigger long-term hydrogen photoproduction in unicellular green algae has two major effects: a decrease in PSII which allows anaerobiosis to be reached and carbohydrate (starch) storage. Starch metabolism has been proposed as one of the major factors of hydrogen production, particularly during the PSII-independent (or indirect) pathway. While starch biosynthesis has been characterized in the green alga Chlamydomonas reinhardtii, little remains known concerning starch degradation. In order to gain a better understanding of starch catabolism pathways and identify those steps likely to limit the starch-dependent hydrogen production, we have designed a genetic screening procedure aimed at isolating mutants of the green alga C. reinhardtii affected in starch mobilization. Using two different screening protocols, the first one based on aerobic starch degradation in the dark and the second one on anaerobic starch degradation in the light, eighteen mutants were isolated among a library of 15,000 insertion mutants, eight (std1-8) with the first screen and ten (sda1-10) with the second. Most of the mutant strains isolated in this study showed a reduction or a delay in the PSII-independent hydrogen production. Further characterization of these mutants should allow the identification of molecular determinants of starch-dependent hydrogen production and supply targets for future biotechnological improvements. (author)

  16. Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant.

    Science.gov (United States)

    Moe, Elin; Rollo, Filipe; Silveira, Célia M; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

    2018-01-05

    Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

    Directory of Open Access Journals (Sweden)

    Kristina M. Ilieva

    2017-09-01

    Full Text Available Monoclonal antibodies find broad application as therapy for various types of cancer by employing multiple mechanisms of action against tumors. Manipulating the Fc-mediated functions of antibodies that engage immune effector cells, such as NK cells, represents a strategy to influence effector cell activation and to enhance antibody potency and potentially efficacy. We developed a novel approach to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies. This entailed coupling single expression vector (pVitro1 antibody cloning, using polymerase incomplete primer extension (PIPE polymerase chain reaction, together with simultaneous Fc region point mutagenesis and high yield transient expression in human mammalian cells. Employing this, we engineered wild type, low (N297Q, NQ, and high (S239D/I332E, DE FcR-binding Fc mutant monoclonal antibody panels recognizing two cancer antigens, HER2/neu and chondroitin sulfate proteoglycan 4. Antibodies were generated with universal mutagenic primers applicable to any IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in small culture volumes, at high yields and within 12 days from design to purified material. Antibody variants conserved their Fab-mediated recognition of target antigens and their direct anti-proliferative effects against cancer cells. Fc mutations had a significant impact on antibody interactions with Fc receptors (FcRs on human NK cells, and consequently on the potency of NK cell activation, quantified by immune complex-mediated calcium mobilization and by antibody-dependent cellular cytotoxicity (ADCC of tumor cells. This strategy for manipulation and testing of Fc region engagement with cognate FcRs can facilitate the design of antibodies with defined effector functions and potentially enhanced efficacy against tumor cells.

  18. Características fisiológicas de microtomateiros fitocromo-mutantes Physiological characteristics of micro-tomato (Lycopersicon esculentum P. Miller) phytochrome-mutants

    National Research Council Canada - National Science Library

    Hyrandir Cabral de Melo; Evaristo Mauro de Castro; Ângela Maria Soares; Cynthia de Oliveira; Sílvio Júnio Ramos

    2009-01-01

    ... (Lycopersicon esculentum P. Miller cv. Micro-Tom) fitocromo-mutantes. A cultivar Micro-Tom e os mutantes aurea (deficiente na biossíntese do cromóforo dos fitocromos), atroviolacea (atv) e high pigment1 (hp1...

  19. AFM images of complexes between amylose and Aspergillus niger glucoamylase mutants, native and mutant starch binding domains: a model for the action of glucoamylase

    DEFF Research Database (Denmark)

    Morris, V. M.; Gunning, A. P.; Faults, C. B.

    2005-01-01

    Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild-type A. niger starch binding domains (SBDS), and mutant SBDs (W563K and W590K) lacking either of the two starch ...

  20. Fitness of Salmonella mutants resistant to antimicrobial peptides.

    Science.gov (United States)

    Lofton, Hava; Anwar, Naeem; Rhen, Mikael; Andersson, Dan I

    2015-02-01

    To examine the effects of mutations in the waaY, phoP and pmrB genes, which confer resistance to antimicrobial peptides (AMPs), on fitness of Salmonella Typhimurium. Survival during low pH, oxidative stress, stationary-phase incubation, exposure to serum and bile and growth in mice and laboratory media were determined by time-kills, disc inhibition assays, competition experiments and optical density measurements. Individual mutations in the waaY gene (involved in LPS core biosynthesis) and in the phoP and pmrB genes (part of two different two-component regulatory systems, phoPQ and pmrAB) conferred no or minor effects on bacterial survival during stressful in vitro conditions or in mice. In contrast, a waaY-phoP-pmrB triple mutant was compromised under most assay conditions. Results from this study show that AMP resistance can be cost-free, as assessed by several assays that attempt to mimic the conditions a bacterium might encounter within a host. Our findings imply that future therapeutic use of AMPs could select for fit mutants with cross-resistance to human defence peptides and that potential resistance development in response to therapeutic use of AMPs needs to be carefully monitored. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes.

    Science.gov (United States)

    Lyozin, George T; Kosaka, Yasuhiro; Bhattacharje, Gourab; Yost, H Joseph; Brunelli, Luca

    2017-04-03

    Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle-driven Enrichment (FPE), a method based on the population genetics "founder principle," to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  2. Nonselective enrichment for yeast adenine mutants by flow cytometry

    Science.gov (United States)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  3. Treatment of MDR1 Mutant Dogs with Macrocyclic Lactones

    Science.gov (United States)

    Geyer, Joachim; Janko, Christina

    2012-01-01

    P-glycoprotein, encoded by the multidrug resistance gene MDR1, is an ATP-driven drug efflux pump which is highly expressed at the blood-brain barrier of vertebrates. Drug efflux of macrocyclic lactones by P-glycoprotein is highly relevant for the therapeutic safety of macrocyclic lactones, as thereby GABA-gated chloride channels, which are confined to the central nervous system in vertebrates, are protected from high drug concentrations that otherwise would induce neurological toxicity. A 4-bp deletion mutation exists in the MDR1 gene of many dog breeds such as the Collie and the Australian Shepherd, which results in the expression of a non-functional P-glycoprotein and is associated with multiple drug sensitivity. Accordingly, dogs with homozygous MDR1 mutation are in general prone to neurotoxicity by macrocyclic lactones due to their increased brain penetration. Nevertheless, treatment of these dogs with macrocyclic lactones does not inevitably result in neurological symptoms, since, the safety of treatment highly depends on the treatment indication, dosage, route of application, and the individual compound used as outlined in this review. Whereas all available macrocyclic lactones can safely be administered to MDR1 mutant dogs at doses usually used for heartworm prevention, these dogs will experience neurological toxicity following a high dose regimen which is common for mange treatment in dogs. Here, we review and discuss the neurotoxicological potential of different macrocyclic lactones as well as their treatment options in MDR1 mutant dogs. PMID:22039792

  4. Neonatal vestibular stimulation and mating in cerebellar mutants.

    Science.gov (United States)

    Guastavino, J M; Larsson, K; Allain, C; Jaisson, P

    1993-05-01

    Two cerebellar mutants, staggerer and reeler, and their congenic nonmutants were used in this experiment. Experimental animals were subjected to intense rotational stimulation on a tilted plane during the first 3 weeks of life, while controls were left nonstimulated. The capacity for mating, as evidenced by vaginal plugs or the occurrence of pregnancy, was assayed during two periods: between 36 and 89 days of age (Experiment A) and between 90 and 120 days of age (Experiment B). During Experiment A the mutants as well as the normals were caged inter se with partners of the opposite sex. During Experiment B the animals were caged with intact, sexually experienced partners. The animals were examined daily for evidence of mating. During Experiment A, only 3 of the 89 couples participating in this study showed evidence of mating. During Experiment B, the number of males of both strains which had mated increased significantly. The staggerer females showed a relatively high level of mating activity, whether stimulated or not. The reeler females, in contrast, rarely mated, although early stimulation significantly increased the level of sexual efficiency. The majority of the normal males and females mated, whether stimulated or not. It was concluded that massive motor-sensory stimulation in infancy, improving gait and body balance in staggerer and reeler mice, may also improve mating efficiency.

  5. Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

    Science.gov (United States)

    Peer, W. A.; Brown, D. E.; Tague, B. W.; Muday, G. K.; Taiz, L.; Murphy, A. S.

    2001-01-01

    Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

  6. Family feud in chemosensitvity: p73 and mutant p53.

    Science.gov (United States)

    Irwin, Meredith S

    2004-03-01

    The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 ("gain of function") are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.

  7. Structural dataset for the PPARγ V290M mutant

    Directory of Open Access Journals (Sweden)

    Ana C. Puhl

    2016-06-01

    Full Text Available Loss-of-function mutation V290M in the ligand-binding domain of the peroxisome proliferator activated receptor γ (PPARγ is associated with a ligand resistance syndrome (PLRS, characterized by partial lipodystrophy and severe insulin resistance. In this data article we discuss an X-ray diffraction dataset that yielded the structure of PPARγ LBD V290M mutant refined at 2.3 Å resolution, that allowed building of 3D model of the receptor mutant with high confidence and revealed continuous well-defined electron density for the partial agonist diclofenac bound to hydrophobic pocket of the PPARγ. These structural data provide significant insights into molecular basis of PLRS caused by V290M mutation and are correlated with the receptor disability of rosiglitazone binding and increased affinity for corepressors. Furthermore, our structural evidence helps to explain clinical observations which point out to a failure to restore receptor function by the treatment with a full agonist of PPARγ, rosiglitazone.

  8. Identification and Characterization of Spontaneous Auxotrophic Mutants in Fusarium langsethiae

    Directory of Open Access Journals (Sweden)

    Olga Gavrilova

    2017-03-01

    Full Text Available Analysis of 49 strains of Fusarium langsethiae originating from northern Europe (Russia, Finland, Sweden, UK, Norway, and Latvia revealed the presence of spontaneous auxotrophic mutants that reflect natural intraspecific diversity. Our investigations detected that 49.0% of F. langsethiae strains were auxotrophic mutants for biotin, and 8.2% of the strains required thiamine as a growth factor. They failed to grow on vitamin-free media. For both prototrophic and auxotrophic strains, no growth defect was observed in rich organic media. Without essential vitamins, a significant reduction in the growth of the auxotrophic strains results in a decrease of the formation of T-2 toxin and diacetoxyscirpenol. In addition, all analysed F. langsethiae strains were distinguished into two subgroups based on PCR product sizes. According to our results, 26 and 23 strains of F. langsethiae belong to subgroups I and II respectively. We determined that the deletion in the intergenic spacer (IGS region of the rDNA of F. langsethiae belonging to subgroup II is linked with temperature sensitivity and causes a decrease in strain growth at 30 °C. Four thiamine auxotrophic strains were found in subgroup I, while 21 biotin auxotrophic strains were detected in subgroups II. To the best of our knowledge, the spontaneous mutations in F. langsethiae observed in the present work have not been previously reported.

  9. Radiometric prescreen for antitumor activity with Saccharomyces cerevisiae mutant strain.

    Science.gov (United States)

    Speedie, M K; Fique, D V; Blomster, R N

    1980-07-01

    After modification, a technique for radiometrically measuring bacterial growth has been applied to a mutant strain of Saccharomyces cerevisiae. The assay is based on inhibition of 14CO2 release from [14C]glucose, which provides an extremely sensitive measure of cellular respiratory activity and growth. The criterion for antitumor activity is the differential inhibition of wild-type and mutant (distorted cell membrane) strains of the yeast. The system was optimized for medium, time of incubation, temperature, and size of inoculum. Known antitumor agents, including bleomycin, actinomycin D, adriamycin, and ellipticine were tested in the system, and differential inhibition was observed. Vincristine showed no inhibitory effects at the concentrations tried. The sensitivity for 20% inhibition ranged from 0.8 micrograms of adriamycin per ml to 0.14 mg of ellipticine per ml. Antifungal agents such as amphotericin B exhibited no differential inhibition. Antibacterial agents were inactive. This method may provide a rapid, sensitive, in vitro quantitative assay for antitumor agents which could be applied to a variety of assay needs and which can be run with facilities and equipment available in most laboratories.

  10. Analysis of Stomatal Patterning in Selected Mutants of MAPK Pathways

    KAUST Repository

    Felemban, Abrar

    2016-05-01

    Stomata are cellular valves in plants that play an essential role in the regulation of gas exchange and are distributed in the epidermis of aerial organs. In Arabidopsis thaliana, stomatal production and development are coordinated by the mitogen-activated protein kinase (MAPK) signalling pathway, which modulates a variety of other processes, including cell proliferation, regulation of cytokinesis, programed cell death, and response to abiotic and biotic stress. The environment also plays a role in stomatal development, by influencing the frequency at which stomata develop in leaves. This thesis presents an analysis of stomatal development in Arabidopsis mutants in two MAPK pathways: MEKK1-MKK1/MKK2-MPK4, and MAP3K17/18-MKK3. Obtained results demonstrate the effect of stress conditions on stomatal development and specify the involvement of analysed MAPK in stomatal patterning. First, both analysed pathways modulate stomatal patterning in Arabidopsis cotyledons. Second, plant growth-promoting bacteria tested enhance stomatal density and affect guard cell morphology. Third, the sucrose or mannitol treatment increases defects in stomatal patterning. Finally, salt stress or high temperature can suppress stomatal defects in mutants of the MEKK1-MKK1/MKK2-MPK4 pathway.

  11. Computational identification of adaptive mutants using the VERT system

    Directory of Open Access Journals (Sweden)

    Winkler James

    2012-04-01

    Full Text Available Background Evolutionary dynamics of microbial organisms can now be visualized using the Visualizing Evolution in Real Time (VERT system, in which several isogenic strains expressing different fluorescent proteins compete during adaptive evolution and are tracked using fluorescent cell sorting to construct a population history over time. Mutations conferring enhanced growth rates can be detected by observing changes in the fluorescent population proportions. Results Using data obtained from several VERT experiments, we construct a hidden Markov-derived model to detect these adaptive events in VERT experiments without external intervention beyond initial training. Analysis of annotated data revealed that the model achieves consensus with human annotation for 85-93% of the data points when detecting adaptive events. A method to determine the optimal time point to isolate adaptive mutants is also introduced. Conclusions The developed model offers a new way to monitor adaptive evolution experiments without the need for external intervention, thereby simplifying adaptive evolution efforts relying on population tracking. Future efforts to construct a fully automated system to isolate adaptive mutants may find the algorithm a useful tool.

  12. Design Methodology - Design Synthesis

    DEFF Research Database (Denmark)

    Andreasen, Mogens Myrup

    2003-01-01

    Design Methodology is part of our practice and our knowledge about designing, and it has been strongly supported by the establishing and work of a design research community. The aim of this article is to broaden the reader¿s view of designing and Design Methodology. This is done by sketching...... the development of Design Methodology through time and sketching some important approaches and methods. The development is mainly forced by changing industrial condition, by the growth of IT support for designing, but also by the growth of insight into designing created by design researchers.......ABSTRACT Design Methodology shall be seen as our understanding of how to design; it is an early (emerging late 60ies) and original articulation of teachable and learnable methodics. The insight is based upon two sources: the nature of the designed artefacts and the nature of human designing. Today...

  13. Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase[C][W][OPEN

    Science.gov (United States)

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-01-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants. PMID:24357602

  14. Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase.

    Science.gov (United States)

    Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

    2014-02-01

    The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

  15. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Directory of Open Access Journals (Sweden)

    Niels H Skotte

    Full Text Available Huntington disease (HD is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs. We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  16. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Science.gov (United States)

    Skotte, Niels H; Southwell, Amber L; Østergaard, Michael E; Carroll, Jeffrey B; Warby, Simon C; Doty, Crystal N; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T; Freier, Susan M; Hung, Gene; Seth, Punit P; Bennett, C Frank; Swayze, Eric E; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  17. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering

    Directory of Open Access Journals (Sweden)

    Nørholm Morten HH

    2010-03-01

    Full Text Available Abstract Background The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR. Results Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering. Conclusions The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.

  18. Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity

    DEFF Research Database (Denmark)

    Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Jers, Carsten

    2017-01-01

    A sialidase (EC 3.2.1.18) from the non-pathogenic Trypanosoma rangeli, TrSA, has been shown to exert trans-sialidase activity after mutation of five specific amino acids in the active site (M96V, A98P, S120Y, G249Y, Q284P) to form the so-called TrSA5mut enzyme. By computational and hypothesis...... driven approaches additional mutations enhancing the trans-sialidase activity have been suggested. In the present work, we made a systematic combination of these mutations leading to seven new variants of the T. rangeli sialidase, having 6-16 targeted amino acid mutations. The resulting enzyme variants...... were analyzed via kinetics for their ability to carry out trans-sialidase reaction using CGMP and D-lactose as substrates. The sialidase variants with 15 and 16 mutations, respectively, exhibited significantly improved trans-sialidase activity for D-lactose sialylation. Our results corroborate...

  19. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    Directory of Open Access Journals (Sweden)

    Juliana Oliveira Lima

    Full Text Available Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

  20. Designing Communication Design

    OpenAIRE

    LØVLIE, Anders Sundnes

    2016-01-01

    Innovating in the field of new media genres requires methods for producing designs that can succeed in being disseminated and used outside of design research labs. This article uses the author's experiences with the development of university courses in communication design to address the research question: How can we design courses to give students the competencies they need to work as designers of new media? Based on existing approaches from UX design and other fields, I present a model that...

  1. Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

    Science.gov (United States)

    Arai, Sayuri; Hayashihara, Kayoko; Kanamoto, Yuki; Shimizu, Kazunori; Hirokawa, Yasutaka; Hanai, Taizo; Murakami, Akio; Honda, Hiroyuki

    2017-08-01

    Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Anne Grünewald

    Full Text Available BACKGROUND: Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD. The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7, as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and

  3. Functional annotation of the mesophilic-like character of mutants in a cold-adapted enzyme by self-organising map analysis of their molecular dynamics.

    Science.gov (United States)

    Fraccalvieri, Domenico; Tiberti, Matteo; Pandini, Alessandro; Bonati, Laura; Papaleo, Elena

    2012-10-01

    Multiple comparison of the Molecular Dynamics (MD) trajectories of mutants in a cold-adapted α-amylase (AHA) could be used to elucidate functional features required to restore mesophilic-like activity. Unfortunately it is challenging to identify the different dynamic behaviors and correctly relate them to functional activity by routine analysis. We here employed a previously developed and robust two-stage approach that combines Self-Organising Maps (SOMs) and hierarchical clustering to compare conformational ensembles of proteins. Moreover, we designed a novel strategy to identify the specific mutations that more efficiently convert the dynamic signature of the psychrophilic enzyme (AHA) to that of the mesophilic counterpart (PPA). The SOM trained on AHA and its variants was used to classify a PPA MD ensemble and successfully highlighted the relationships between the flexibilities of the target enzyme and of the different mutants. Moreover the local features of the mutants that mostly influence their global flexibility in a mesophilic-like direction were detected. It turns out that mutations of the cold-adapted enzyme to hydrophobic and aromatic residues are the most effective in restoring the PPA dynamic features and could guide the design of more mesophilic-like mutants. In conclusion, our strategy can efficiently extract specific dynamic signatures related to function from multiple comparisons of MD conformational ensembles. Therefore, it can be a promising tool for protein engineering.

  4. Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Hassan Abdel-Latif A. Mohamed

    2006-06-01

    Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

  5. Mutant pso8-1 of Saccharomyces cerevisiae, sensitive to photoactivated psoralens, UV radiation, and chemical mutagens, contains a rad6 missense mutant allele.

    Science.gov (United States)

    Rolla, H; Grey, M; Schmidt, C L; Niegemann, E; Brendel, M; Henriques, J A P

    2002-07-01

    A novel mutant isolate of Saccharomyces cerevisiae, sensitive to photoactivated mono- and bi-functional psoralens, to UV at 254 nm (UVC), and to nitrosoguanidine, was found to complement the photoactivated psoralen-sensitivity phenotype conferred by the pso1- pso7 mutations and was therefore named pso8-1. A constructed pso8-1 rad4-4 double mutant was super-sensitive to UVC, thus indicating a synergistic interaction of the two mutant alleles. Molecular cloning via complementation of the pso8 mutant's sensitivity phenotype and genetic studies revealed that pso8 is allelic to RAD6. While a pso8-1 mutant had low mutagen-induced mutability, homoallelic diploids showed nearly wild-type sporulation. Sequence analysis of the mutant allele showed pso8-1 to contain a novel, hitherto undescribed T-->C transition in nucleotide position 191, leading to a substitution by leucine of a highly conserved proline at position 64, Rad6-[P64L], which may have severe consequences for the tertiary structure (and hence binding to Rad18p) of the mutant protein.

  6. Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.

    Science.gov (United States)

    Pourebrahim, Rasoul; Zhang, Yun; Liu, Bin; Gao, Ruli; Xiong, Shunbin; Lin, Patrick P; McArthur, Mark J; Ostrowski, Michael C; Lozano, Guillermina

    2017-09-15

    TP53 is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single p53 mutation (p53R172H) or p53 loss in osteoblasts. Our study confirmed that p53 mutant mice developed osteosarcomas with increased metastasis as compared with p53-null mice. Comprehensive transcriptome RNA sequencing (RNA-seq) analysis of 16 tumors identified a cluster of small nucleolar RNAs (snoRNAs) that are highly up-regulated in p53 mutant tumors. Regulatory element analysis of these deregulated snoRNA genes identified strong enrichment of a common Ets2 transcription factor-binding site. Homozygous deletion of Ets2 in p53 mutant mice resulted in strong down-regulation of snoRNAs and reversed the prometastatic phenotype of mutant p53 but had no effect on osteosarcoma development, which remained 100% penetrant. In summary, our studies identify Ets2 inhibition as a potential therapeutic vulnerability in p53 mutant osteosarcomas. © 2017 Pourebrahim et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice

    Directory of Open Access Journals (Sweden)

    Dickson Dennis W

    2011-10-01

    model remain unknown. However, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity. The mutant model will serve as a valuable tool in the development of future studies designed to uncover pathways associated with TDP-43 neurotoxicity and the precise roles TDP-43 RNA targets play in neurodegeneration.

  8. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice

    Science.gov (United States)

    Leushacke, Marc; Li, Ling; Wong, Julin S.; Chiam, Poh Cheang; Rahmat, Siti Aishah Binte; Mann, Michael B.; Mann, Karen M.; Barker, Nick; Lozano, Guillermina; Terzian, Tamara; Lane, David P.

    2015-01-01

    The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy. PMID:26255629

  9. Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia).

    Science.gov (United States)

    Jain, D K; Bordeleau, L M

    1990-12-01

    Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif (r) showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.

  10. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

    Science.gov (United States)

    Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

    1995-04-01

    An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants.

  11. Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus

    Energy Technology Data Exchange (ETDEWEB)

    Pierson, B.K.; Keith, L.M.; Leovy, J.G.

    1984-07-01

    Mutants deficient in the production of bateriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus, Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions. 11 references, 4 figures, 3 tables.

  12. Novel polyketide metabolites from Streptomyces rimosus mutant strain R1059.

    Science.gov (United States)

    Deseo, Myrna A; Hunter, lain S; Waterman, Peter G

    2005-12-01

    Three novel polyketide metabolites were isolated from laboratory-scale fermentation of the Streptomyces rimosus mutant strain R1059. Structural elucidation of the compounds was based on NMR experiments. The compounds were characterized as naphthalene derivatives: (rel)-4beta,8-dihydroxy-3alpha-hydroxymethyl-4alpha-methyl-1,2,3,4-tetrahydronaphthalene1-one (1), 4xi8-dihydroxy-3-hydroxymethyl-4xi-methyl-1,4-dihydronaphthalene-1-one (2) and (rel)-4beta,8-dihydroxy-3alpha-O-[alpha-glucopyranosyl]hydroxymethyl-4alpha-methyl-1,2,3,4-tetrahydronaphthalene-1-one (3). The compounds isolated appear to be derived via a shorter polyketide backbone than oxytetracycline (4), the normal end-product made by the parent of this strain. Compound 3 was the glucoside of 1 and must be formed as a post-PKS reaction by the activation of a glycosyl transferase, which has not been reported in this species before.

  13. Induction of Male sterile mutants in vegetable crops

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Nobuhiko (Hokkaido National Agricultural Experiment Station, Sapporo (Japan))

    1982-03-01

    The cultivars of vegetable crops in Japan are almost all F/sub 1/ hybrid lines. These hybrid cultivars are superior in yield, quality and uniformity by heterosis, and play an important role in the protection of breeder's rights. Utilization of male sterile mutants has such advantages as the reduction of cost for F/sub 1/ production by saving labor, production of better seeds, that is, pollination without emasculation and avoidance of contamination caused by self pollination. Male sterility must be used for some species in which seed production is difficult because of tiny flowers and meager seed production by artificial crossing such as carrot and onion, and those in which pollination by bag or emasculation is expensive such as tomato, and sweet pepper. However, for vegetable crop breeeding, the induction and use of genetic male sterility are more difficult than for other crops, considering the economy and efficiency of research because the type of cultivars needed changes rapidly.

  14. Characterization Of Laccase T-DNA Mutants In Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Andersen, Jeppe R; Asp, Torben; Mansfield, Shawn

    Laccases (P-diphenol:O2 oxidoreductase; EC 1.10.3.2), also termed laccase-like multicopper oxidases, are blue copper-containing oxidases which comprise multigene families in plants. In the Arabidopsis thaliana genome, 17 laccase genes (LAC1 to LAC17) have been annotated. To identify laccases...... involved in cell wall biosynthesis in Arabidopsis primary stems we have developed homozygous T-DNA mutants for 14 individual laccases. Six laccases are highly expressed in the wild type primary stem, four of which (LAC2, LAC4, LAC12, and LAC17) show correlated gene expression with one to several genes (e...... different and distinct biochemical pathways and that laccases might be involved in polymerization of both polysaccharides and monolignols in the Arabidopsis cell wall....

  15. Longevity mutants do not establish any "new science" of ageing.

    Science.gov (United States)

    Holliday, Robin; Rattan, Suresh I S

    2010-08-01

    The biological reasons for ageing are now well known, so it is no longer an unsolved problem in biology. Furthermore, there is only one science of ageing, which is continually advancing. The significance and importance of the mutations that lengthen the lifespan of invertebrates can be assessed only in relationship to previous well-established studies of ageing. The mutant strains of model organisms that increase longevity have altered nutrient signalling pathways similar to the effects of dietary restriction, and so it is likely that there is a shift in the trade-off between reproduction and maintenance of the soma. To believe that the isolation and characterisation of a few invertebrate mutations (as well as those in yeast) will "galvanise" the field and provide new insights into human ageing is an extreme point of view which does not recognize the huge progress in ageing research that has been made in the last 50 years or so.

  16. Validating regulatory predictions from diverse bacteria with mutant fitness data.

    Directory of Open Access Journals (Sweden)

    Shiori Sagawa

    Full Text Available Although transcriptional regulation is fundamental to understanding bacterial physiology, the targets of most bacterial transcription factors are not known. Comparative genomics has been used to identify likely targets of some of these transcription factors, but these predictions typically lack experimental support. Here, we used mutant fitness data, which measures the importance of each gene for a bacterium's growth across many conditions, to test regulatory predictions from RegPrecise, a curated collection of comparative genomics predictions. Because characterized transcription factors often have correlated fitness with one of their targets (either positively or negatively, correlated fitness patterns provide support for the comparative genomics predictions. At a false discovery rate of 3%, we identified significant cofitness for at least one target of 158 TFs in 107 ortholog groups and from 24 bacteria. Thus, high-throughput genetics can be used to identify a high-confidence subset of the sequence-based regulatory predictions.

  17. Boc modifies the holoprosencephaly spectrum of Cdo mutant mice

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2011-05-01

    Holoprosencephaly (HPE is caused by a failure to form the midline of the forebrain and/or midface. It is one of the most common human birth defects, but clinical expression is extremely variable. HPE is associated with mutations in the sonic hedgehog (SHH pathway. Mice lacking the Shh pathway regulator Cdo (also called Cdon display HPE with strain-dependent penetrance and expressivity, implicating silent modifier genes as one cause of the variability. However, the identities of potential HPE modifiers of this type are unknown. We report here that whereas mice lacking the Cdo paralog Boc do not have HPE, Cdo;Boc double mutants on a largely Cdo-resistant genetic background have lobar HPE with strong craniofacial anomalies and defects in Shh target gene expression in the developing forebrain. Boc is therefore a silent HPE modifier gene in mice. Furthermore, Cdo and Boc have specific, selective roles in Shh signaling in mammals, because Cdo;Boc double-mutant mice do not display the most severe HPE phenotype seen in Shh-null mice, nor do they have major defects in digit patterning or development of vertebrae, which are also Shh-dependent processes. This is in contrast to reported observations in Drosophila, where genetic removal of the Cdo and Boc orthologs Ihog and Boi results in a complete loss of response to the hedgehog ligand. Therefore, there is evolutionary divergence between mammals and insects in the requirement of the hedgehog pathway for Cdo/Ihog family members, with mammalian development involving additional factors and/or distinct mechanisms at this level of pathway regulation.

  18. HCN channels in the heart: lessons from mouse mutants.

    Science.gov (United States)

    Herrmann, S; Hofmann, F; Stieber, J; Ludwig, A

    2012-05-01

    Hyperpolarization-activated cation channels generate the I(f) current in the heart. In the sino-atrial node (SAN), I(f) is thought to play an essential role in setting the heart rate and mediating its autonomic control. This review focuses on the role of I(f) in pacemaking and non-pacemaking cardiomyocytes and the resulting therapeutic implications. HCN4 represents the principal isoform underlying sino-atrial I(f) , but other isoforms may also be of importance. To examine the functional role of cardiac channels, several mouse mutants, most of them targeting HCN4, have been generated by different groups. Unexpectedly, these lines display greatly different and as yet unexplained phenotypes. We provide an overview about these HCN mutants and suggest an interpretation of the functional significance of I(f) in the SAN in light of these studies. HCN channels are also present in ventricular myocytes, and an up-regulation of I(f) in the hypertrophic and failing heart may contribute to arrhythmogenesis. Inhibition of I(f) by HCN channel blockers is a novel approach in the treatment of cardiac disorders, and ivabradine is approved for treatment of stable angina pectoris. Remarkably, a recent clinical trial assessing this substance in heart failure showed a significantly improved outcome. The mechanism underlying this beneficial effect is not yet clear and might lie beyond heart rate slowing. Thus, the growing knowledge about cardiac HCN channels will undoubtedly promote the development of the promising class of HCN channel blockers. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  19. [Establishment of a mutant Lumican transgenic mouse model].

    Science.gov (United States)

    Song, Yanzheng; Zhao, Yanyan; Zhang, Fengju; Yu, Yanqiu; Ma, Ling

    2014-01-01

    Pathological myopia (PM) is a hereditary ocular disease leading to severe loss of visual acuity and blindness. Lumican gene (LUM) is one of those candidate genes of PM. The purpose of this study was to establish a mutant Lumican transgenic mouse model, and to prepare for the further study of the pathogenesis of PM. Experimental study. Mutation of LUM gene was created by site-directed mutagenesis. Recombinant DNA techniques were used for the construction of the pRP. EX3d-EF1A>LUM/flag>IRES/hrGFP transgene. The gene fragments were microinjected into the zygote male pronuclei of BDF1 mice, and then the zygote cells alive were transplanted into the oviduct of acceptor pregnant female ICR mice. The F0 generation transgenic mice obtained were named C57-TgN (LUM)CCMU. Genome DNA from mice tail was detected by PCR and Western blotting. Six of 31 F0 generation mice were positive transgenic mice. The western blotting study showed that the flag-tag was expressed in the mouse tail tissue. Sixty-eight of 128 mice (F1 to F3 generation) were positive transgenic mice, the positive rate is 53.13%. The mutant Lumican (cDNA 596T>C) transgenic mouse model has been established. This model will provide fundamental conditions for studies of the pathogenesis of PM. Also it will be the basis of further studies about the effect of Lumican mutation on the development of PM and structure and function of the extra cellular matrix.

  20. Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

    Science.gov (United States)

    Mosenson, Jeffrey A.; Zloza, Andrew; Nieland, John D.; Garrett-Mayer, Elizabeth; Eby, Jonathan M.; Huelsmann, Erica J.; Kumar, Previn; Denman, Cecele J.; Lacek, Andrew T.; Kohlhapp, Frederick J.; Alamiri, Ahmad; Hughes, Tasha; Bines, Steven D.; Kaufman, Howard L.; Overbeck, Andreas; Mehrotra, Shikhar; Hernandez, Claudia; Nishimura, Michael I.; Guevara-Patino, Jose A.; Le Poole, I. Caroline

    2013-01-01

    Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo. PMID:23447019

  1. Glycine receptor mouse mutants: model systems for human hyperekplexia.

    Science.gov (United States)

    Schaefer, Natascha; Langlhofer, Georg; Kluck, Christoph J; Villmann, Carmen

    2013-11-01

    Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations. © 2013 The British Pharmacological Society.

  2. A mutant gene that increases gibberellin production in Brassica

    Energy Technology Data Exchange (ETDEWEB)

    Rood, S.B. (Univ. of Lethbridge, Alberta (Canada)); Williams, P.H. (Univ. of Wisconsin, Madison (USA)); Pearce, D.; Pharis, R.P. (Univ. of Calgary, Alberta (Canada)); Murofushi, Noboru (Univ. of Tokyo (Japan)); Mander, L.N. (Australian National Univ., Canberra (Australia))

    1990-07-01

    A single gene mutant (elongated internode (ein/ein)) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A{sub 3} (GA{sub 3}) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA{sub 1} and GA{sub 3} were estimated by gas chromatography-selected ion monitoring using ({sup 2}H)GA{sub 1} as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA{sub 20} and GA{sub 1}, and the rate of GA{sub 19} metabolism were simultaneously analyzed. Levels of GA{sub 1} and GA{sub 20} were 4.6- and 12.9-fold higher, respectively, and conversions to GA{sub 20} and GA{sub 1} were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA{sub 1} biosynthesis in ein, the conversion of ({sup 3}H)GA{sub 20} to ({sup 3}H) GA{sub 1} was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA{sub 1} biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A{sub 1} and A{sub 3}.

  3. Transcriptome Analysis of a New Peanut Seed Coat Mutant for the Physiological Regulatory Mechanism Involved in Seed Coat Cracking and Pigmentation.

    Science.gov (United States)

    Wan, Liyun; Li, Bei; Pandey, Manish K; Wu, Yanshan; Lei, Yong; Yan, Liying; Dai, Xiaofeng; Jiang, Huifang; Zhang, Juncheng; Wei, Guo; Varshney, Rajeev K; Liao, Boshou

    2016-01-01

    Seed-coat cracking and undesirable color of seed coat highly affects external appearance and commercial value of peanuts ( Arachis hypogaea L.). With an objective to find genetic solution to the above problems, a peanut mutant with cracking and brown colored seed coat (testa) was identified from an EMS treated mutant population and designated as "peanut seed coat crack and brown color mutant line ( pscb )." The seed coat weight of the mutant was almost twice of the wild type, and the germination time was significantly shorter than wild type. Further, the mutant had lower level of lignin, anthocyanin, proanthocyanidin content, and highly increased level of melanin content as compared to wild type. Using RNA-Seq, we examined the seed coat transcriptome in three stages of seed development in the wild type and the pscb mutant. The RNA-Seq analysis revealed presence of highly differentially expressed phenylpropanoid and flavonoid pathway genes in all the three seed development stages, especially at 40 days after flowering (DAF40). Also, the expression of polyphenol oxidases and peroxidase were found to be activated significantly especially in the late seed developmental stage. The genome-wide comparative study of the expression profiles revealed 62 differentially expressed genes common across all the three stages. By analyzing the expression patterns and the sequences of the common differentially expressed genes of the three stages, three candidate genes namely c36498_g1 (CCoAOMT1), c40902_g2 (kinesin) , and c33560_g1 (MYB3) were identified responsible for seed-coat cracking and brown color phenotype. Therefore, this study not only provided candidate genes but also provided greater insights and molecular genetic control of peanut seed-coat cracking and color variation. The information generated in this study will facilitate further identification of causal gene and diagnostic markers for breeding improved peanut varieties with smooth and desirable seed coat color.

  4. Transcriptome analysis of a new peanut seed coat mutant for the physiological regulatory mechanism involved in seed coat cracking and pigmentation

    Directory of Open Access Journals (Sweden)

    Liyun Wan

    2016-10-01

    Full Text Available Seed-coat cracking and undesirable color of seed coat highly affects external appearance and commercial value of peanuts (Arachis hypogaea L.. With an objective to find genetic solution to the above problems, a peanut mutant with cracking and brown colored seed coat (testa was identified from an EMS treated mutant population and designated as peanut seed coat crack and brown color mutant line (pscb. The seed coat weight of the mutant was almost twice of the wild type, and the germination time was significantly lower than wild type. Further, the mutant had lower level of lignin, anthocyanin, proanthocyandin content and highly increased level of melanin content as compared to wild type. Using RNA-Seq, we examined the seed coat transcriptome in three stages of seed development in the wild type and the pscb mutant. The RNA-Seq analysis revealed presence of highly differentially expressed phenylpropanoid and flavonoid pathway genes in all the three seed development stages, especially at 40 days after flowering (DAF40. Also, the expression of polyphenol oxidases and peroxidase were found to be activated significantly especially in the late seed developmental stage. The genome-wide comparative study of the expression profiles revealed 62 differentially expressed genes common across all the three stages. By analyzing the expression patterns and the sequences of the common differentially expressed genes of the three stages, three candidate genes namely c36498_g1 (CCoAOMT1, c40902_g2 (kinesin and c33560_g1 (MYB3 were identified responsible for seed-coat cracking and brown color phenotype. Therefore, this study not only provided candidate genes but also provided greater insights and molecular genetic control of peanut seed-coat cracking and color variation. The information generated in this study will facilitate further identification of causal gene and diagnostic markers for breeding improved peanut varieties with smooth and desirable seed coat color.

  5. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

    Directory of Open Access Journals (Sweden)

    Victoria A Lawson

    Full Text Available BACKGROUND: The accumulation of protease resistant conformers of the prion protein (PrP(res is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. METHODOLOGY/PRINCIPAL FINDING: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS from the PrP(C substrate was found to specifically prevent PrP(res formation seeded by mouse derived PrP(Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res formation, while having no effect on PrP(res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. CONCLUSIONS/SIGNIFICANCE: Cofactor requirements for PrP(res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

  6. Burkholderia thailandensis oacA mutants facilitate the expression of Burkholderia mallei-like O polysaccharides.

    Science.gov (United States)

    Brett, Paul J; Burtnick, Mary N; Heiss, Christian; Azadi, Parastoo; DeShazer, David; Woods, Donald E; Gherardini, Frank C

    2011-02-01

    Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates.

  7. Burkholderia thailandensis oacA Mutants Facilitate the Expression of Burkholderia mallei-Like O Polysaccharides▿

    Science.gov (United States)

    Brett, Paul J.; Burtnick, Mary N.; Heiss, Christian; Azadi, Parastoo; DeShazer, David; Woods, Donald E.; Gherardini, Frank C.

    2011-01-01

    Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates. PMID:21115721

  8. Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Rozpedowska, E.; Le Breton, C.

    2007-01-01

    to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced...... that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution....

  9. Filtration enrichment method for isolation of auxotrophic mutants of Trichoderma harzianum rifai

    Directory of Open Access Journals (Sweden)

    Cassiolato Ana Maria R.

    1999-01-01

    Full Text Available The isolation of genetic markers, like drug resistance and auxotrophy, is a laborious but important step in genetic research. The isolation of auxotrophic mutants of Trichoderma harzianum using the filtration enrichment technique was more effective than using the total isolation technique. Most of 12 auxotrophic mutants exhibited similar growth rate and higher sporulation when compared with the wild type, but only two mutants (TWS-410 and TW5-523 could grow in 500µg/L of benomyl.

  10. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    OpenAIRE

    Lima,Juliana de Oliveira; Pereira, Jorge Fernando; Araújo,Elza Fernandes; Queiroz, Marisa Vieira de

    2017-01-01

    Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also stud...

  11. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    OpenAIRE

    Lima,Juliana de Oliveira; Pereira, Jorge Fernando; Araújo,Elza Fernandes; Queiroz, Marisa Vieira de

    2017-01-01

    Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were ...

  12. Mutant p53 promotes cell spreading and migration via ARHGAP44.

    Science.gov (United States)

    Xu, Jinjin; Jiao, Jian; Xu, Wei; Ji, Lei; Jiang, Dongjie; Xie, Shaofang; Kubra, Syeda; Li, Xiaotao; Fu, Junjiang; Xiao, Jianru; Zhang, Bianhong

    2017-09-01

    The tumor suppressor p53 protein is either lost or mutated in about half of all human cancers. Loss of p53 function is well known to influence cell spreading, migration and invasion. While expression of mutant p53 is not equivalent to p53 loss, mutant p53 can acquire new functions to drive cell spreading and migration via different mechanisms. In our study, we found that mutant p53 significantly increased cell spreading and migration when comparing with p53-null cells. RNA-Seq analysis suggested that Rho GTPase activating protein 44 (ARHGAP44) is a new target of mutant p53, which suppressed ARHGAP44 transcription. ARHGAP44 has GAP activity and catalyze GTP hydrolysis on Cdc42. Higher level of GTP-Cdc42 was correlated with increase expression of mutant p53 and reduced ARHGAP44. Importantly, wt-ARHGAP44 but not mutant ARHGAP44 (R291A) suppressed mutant p53 mediated cell spreading and migration. Bioinformatics analysis indicated lower expression of ARHGAP44 in lung carcinoma compared with normal tissues, which was verified by RT-qPCR using specimens from patients. More interestingly, ARHGAP44 mRNA level was lower in tumors with mutant p53 than those with normal p53. Collectively, our results disclose a new mechanism by which mutant p53 stimulates cell spreading and migration.

  13. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M. (Universite du Quebec a Montreal (Canada))

    1989-10-01

    The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population.

  14. The Electrogenic Bacterium Shewanella Oneidensis MR-1 and its Mutants with Increased Reducing Capacity

    Science.gov (United States)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Mordkovich, N. N.; Shakulov, R. S.; Debabov, V. G.

    2013-02-01

    Mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited an increased reducing activity and a higher rate of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S.oneidensis MR-1 will be used in microbial fuel cells (MFC) to enhance energy production from organic compounds. The strain S. oneidensis MR-1 and its mutants with an increased electron production will be used as a good source of bioelectricity in MFC in the experiments on the International Space Station.

  15. Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii.

    Science.gov (United States)

    Wang, Dan; Wang, Yuan Chun; Wu, Li Juan; Liu, Jian Xin; Zhang, Pan; Jiao, Jian; Yan, Hui; Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2016-03-01

    Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0-30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis.

  16. Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor.

    Science.gov (United States)

    Lambert, Stéphany; Traxler, Matthew F; Craig, Matthias; Maciejewska, Marta; Ongena, Marc; van Wezel, Gilles P; Kolter, Roberto; Rigali, Sébastien

    2014-08-01

    Streptomyces coelicolor is an important model organism for developmental studies of filamentous GC-rich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with bldA, bldJ, and ptsH mutants severely impaired in DFO production, while bldF, bldK, crr and ptsI mutants overproduce DFO. Morphogenesis of bldK and bldJ mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldJ mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldK mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthens the role of siderophores and iron acquisition in morphological development of actinomycetes.

  17. Spontaneous alternation, motor activity, and spatial learning in hot-foot mutant mice.

    Science.gov (United States)

    Filali, M; Lalonde, R; Bensoula, A N; Guastavino, J M; Lestienne, F

    1996-01-01

    Hot-foot mutant mice, characterized by defective innervation of Purkinje cells and an ataxic gait, were less active than normal mice in a T-maze. In spontaneous alternation testing with either single or multiple trials, hot-foot mutants, contrary to normal mice, did not alternate above chance. Moreover, the mutants had a higher number of errors and higher escape latencies in a water-filled Z-maze. These results indicate that in addition to motor coordination deficits, these cerebellar mutants have deficits in spatial learning and perseverate choices of maze arms.

  18. A comparison of direct infusion MS and GC-MS for metabolic footprinting of yeast mutants

    DEFF Research Database (Denmark)

    Mass, S.; Villas-Bôas, Silas Granato; Hansen, Michael Adsetts Edberg

    2007-01-01

    with glucose as the carbon source. The mutants evaluated were cat8, gln3, ino2, opi1, and nil1, all with deletion of genes involved in nutrient sensing and regulation. From the analysis, we found that both methods can be used to classify mutants, but the classification depends on which metabolites are measured....... Thus, the GC-MS method is good for classification of mutants with altered nitrogen regulation as it primarily measures amino acids, whereas this method cannot classify mutants involved in regulation of phospholipids metabolism as well as the direct infusion MS (DI-MS) method. From the analysis, we find...

  19. Caenorhabditis elegans mutants having altered preference of chemotaxis behavior during simultaneous presentation of two chemoattractants.

    Science.gov (United States)

    Lin, Lin; Wakabayashi, Tokumitsu; Oikawa, Tomohiro; Sato, Tsutomu; Ogurusu, Tarou; Shingai, Ryuzo

    2006-11-01

    Upon presentation of two distinct chemoattractants such as sodium acetate and diacetyl simultaneously, the nematode Caenorhabditis elegans was preferentially attracted by one of these chemoattractants. We isolated two mutants having altered preference of chemotaxis behavior toward simultaneous presentation of sodium acetate and diacetyl. The chep-1(qr1) (CHEmosensory Preference) mutant preferred sodium acetate to diacetyl, while the chep-2(qr2) mutant preferred diacetyl to sodium acetate in simultaneous presentation of these chemoattractants. The chemotaxis behavior of chep-2(qr2) mutant in simultaneous presentation suggests a function of chep-2 gene products within the chemosensory informational integration pathway as well as in the chemosensory pathway.

  20. Membrane function in lipid mutants of Arabidopsis. First year progress report

    Energy Technology Data Exchange (ETDEWEB)

    Browse, J.A.

    1993-06-01

    Progress on the biochemical characterization of the fad3 mutants deficient in 18:3 fatty acid synthesis and the fab2 mutant that accumulates increased amounts of 18:0 is described. Studies of the cell biology and physiology of the fab2 and fad2 mutants have provided evidence for some of the critical roles played by unsaturated fatty acids as components of plant membranes. Finally, the fab2 mutant has allowed us to carry out the first isolation and characterization of intergenic suppressor mutations in a higher plant.

  1. Mutantes morfológicos de algodoeiro herbáceo como fonte de resistência ao bicudo Morphological mutants of upland cotton as source of boll weevil resistance

    Directory of Open Access Journals (Sweden)

    Francisco das Chagas Vidal Neto

    2005-02-01

    Full Text Available Este trabalho teve como objetivo avaliar os efeitos de três características morfológicas mutantes de linhagens de algodoeiro herbáceo (Gossypium hirsutum L. r. latifolium Hutch., isoladas ou combinadas no mesmo genótipo, como fonte de resistência ao bicudo, Anthonomus grandis Boheman, 1843 (Coleoptera, Curculionidae. O experimento foi conduzido em campo, sob infestação natural, com delineamento de blocos ao acaso e arranjo fatorial 2´3 com um tratamento adicional, com quatro repetições. Em teste com chance de escolha, a característica bráctea frego foi a que apresentou maior redução no dano de oviposição pelo bicudo (34,71%, em relação ao equivalente normal. A folha "okra" reduziu o dano apenas quando associada à bráctea frego (40%. A combinação das três características mutantes na mesma planta proporcionou a menor porcentagem de botões com dano de oviposição (23,13%.This work aimed to evaluate the effects of three morphological mutants of upland cotton lines (Gossypium hirsutm L. r. latifolium Hutch., isolated or in combination in the same cotton genotype, as a source of resistance to boll weevil, Anthonomus grandis Boheman, 1843 (Coleoptera, Curculionidae. The experiment was carried out in the field, under natural infestation, with a completely randomized block design arranged in a factorial 2´3 plus an additional treatment, with four replications. In a multiple choice test, the character mutant frego bract presented the higher reduction on boll weevil oviposition damage (34.71%, in relation to the normal equivalent. The okra leaf reduced the boll weevil damage only when associated with frego bract (40%. The combination of the three mutant characters in the same plant presented the least square percent with oviposition damage (23.13%.

  2. Designing Communication Design

    DEFF Research Database (Denmark)

    Løvlie, Anders Sundnes

    2016-01-01

    Innovating in the field of new media genres requires methods for producing designs that can succeed in being disseminated and used outside of design research labs. This article uses the author's experiences with the development of university courses in communication design to address the research...... question: How can we design courses to give students the competencies they need to work as designers of new media? Based on existing approaches from UX design and other fields, I present a model that has demonstrated its usefulness in the development of commercial products and services. The model...

  3. The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast.

    Science.gov (United States)

    Ito, K; Ebihara, K; Nakamura, Y

    1998-08-01

    Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3. The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis. In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1. The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35). All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids. To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae. These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.

  4. Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation.

    Science.gov (United States)

    Roth, W W; Bragg, P W; Corrias, M V; Reddy, N S; Dholakia, J N; Wahba, A J

    1987-01-01

    The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000. During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome. To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene. We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp. eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp. eEF-Tu. From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts. All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu. We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays. A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells. The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds. A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei. Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA. In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu. The derived amino acid sequence is compared with sequences from other eucaryotes. Images PMID:3481036

  5. Designing Material Materialising Design

    DEFF Research Database (Denmark)

    Nicholas, Paul

    2013-01-01

    and operational models by which to specify and materialise causal relationships between configuration and transformation, these investigations reveal a new locus for architectural instruction that requires new kinds of design information, new representational models, and different modes of design control.......Designing Material Materialising Design documents five projects developed at the Centre for Information Technology and Architecture (CITA) at the Royal Danish Academy of Fine Arts, School of Architecture. These projects explore the idea that new designed materials might require new design methods....... Focusing on fibre reinforced composites, this book sustains an exploration into the design and making of elastically tailored architectural structures that rely on the use of computational design to predict sensitive interdependencies between geometry and behaviour. Developing novel concepts...

  6. How Expert Designers Design

    NARCIS (Netherlands)

    Dr. Peter Sloep; J. van Merrienboer; C. Carr; P. Kirschner

    2003-01-01

    This paper discusses two studies - the one in a business context, the other in a university context - carried out with expert educational designers. The studies aimed to determine the priorities experts claim to employ when designing competence-based learning environments. Designers in both contexts

  7. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    Energy Technology Data Exchange (ETDEWEB)

    Gindt, Yvonne Marie [Univ. of California, Berkeley, CA (United States)

    1993-04-01

    The role of the Lcm (I), β18 (II), and αAP-B (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, Amax = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  8. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    Energy Technology Data Exchange (ETDEWEB)

    Gindt, Y.M.

    1993-04-01

    The role of the L[sub cm] (I), [beta][sup 18] (II), and [alpha][sup AP-B] (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A[sub max] = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  9. Easily accessible polycyclic amines that inhibit the wild-type and amantadine-resistant mutants of the M2 channel of influenza A virus.

    Science.gov (United States)

    Rey-Carrizo, Matias; Barniol-Xicota, Marta; Ma, Chunlong; Frigolé-Vivas, Marta; Torres, Eva; Naesens, Lieve; Llabrés, Salomé; Juárez-Jiménez, Jordi; Luque, Francisco J; DeGrado, William F; Lamb, Robert A; Pinto, Lawrence H; Vázquez, Santiago

    2014-07-10

    Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.

  10. Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants.

    Science.gov (United States)

    Capra, M L; Mercanti, D J; Rossetti, L C; Reinheimer, J A; Quiberoni, A

    2011-08-01

    To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC-A. Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD-PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage-resistant mutants were tested against phages PL-1, J-1, A2 and MLC-A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC-A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC-A but also to other Lact. casei and Lact. paracasei phages. This study highlights isolation of spontaneous bacteriophage-resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  11. A Nearly Non-Functional Mutant Allele of the Storage Protein Locus Hor2 in Barley

    DEFF Research Database (Denmark)

    Doll, Hans

    1980-01-01

    The low content of the storage protein fraction hordein-2 in the high-lysine mutant Risø 56 is due to a mutation at or near the locus Hor2 coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect on the electrophoretic banding pattern of hordein-2, but co-dominant...

  12. Ethanol metabolism in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Sulter, G.J.; Klei, I.J. van der; Schanstra, J.P.; Harder, W.; Veenhuis, M.

    1991-01-01

    This paper describes ethanol metabolism in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. The PER mutant was able to use ethanol as sole-carbon source but showed reduced growth rates compared to wild-type cells together with a reduced rate of ethanol utilization under µmax conditions.

  13. Bridging the gap between chemistry, physiology, and evolution: Quantifying the functionality of sperm whale myoglobin mutants

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Kepp, Kasper Planeta

    2011-01-01

    This work merges a large set of previously reported thermochemical data for myoglobin (Mb) mutants with a physiological model of O2-transport and -storage. The model allows a quantification of the functional proficiency of myoglobin (Mb) mutants under various physiological conditions, i.e. O2-con...

  14. In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB

    DEFF Research Database (Denmark)

    Hediger, Martin Robert; De Vico, Luca; Rannes, Julie Bille

    2013-01-01

    Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., 2012) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barrie...

  15. Lipase production from a wild (LPF-5) and a mutant (HN1) strain of ...

    African Journals Online (AJOL)

    Lipase production from a wild (LPF-5) and a mutant (HN1) strain of Aspergillus niger. Arun Kumar Sharma, Vinay Sharma, Jyoti Saxena, Arindam Kuila. Abstract. In this study, a wild (LPF-5) and a mutant (HN1) strain of A. niger were compared for lipase production. Several physical parameters (carbon source, nitrogen ...

  16. A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

    Science.gov (United States)

    Witherow, D. Scott

    2016-01-01

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

  17. Biofilm formation by exopolysaccharide mutants of Leuconostoc mesenteroides strain NRRL B-1355

    Science.gov (United States)

    Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. A set of mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces ...

  18. A Laboratory Exercise for Isolation and Characterizing Microbial Mutants with Metabolic Defects.

    Science.gov (United States)

    Doe, Frank J.; Leslie, John F.

    1993-01-01

    Describes science experiments for undergraduate biology instruction on the concepts of mutation and characterization of the resulting mutant strains. The filamentous fungi "Fusarium moniliforme" is used to illustrate the induction of mutants (mutagenesis), identification of the mutated gene, construction of a biochemical pathway, and…

  19. Comparison of arabidopsis stomatal density mutants indicates variation in water stress responses and potential epistatic effects

    Science.gov (United States)

    Shaneka S. Lawson; Paula M. Pijut; Charles H. Michler

    2014-01-01

    Recent physiological analysis of Arabidopsis stomatal density (SD) mutants indicated that SD was not the major factor controlling aboveground biomass accumulation. Despite the general theory that plants with fewer stomata have limited biomass acquisition capabilities, epf1 and several other Arabidopsis mutants varied significantly in leaf fresh...

  20. Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold

    Energy Technology Data Exchange (ETDEWEB)

    Kalayanamitr, A.; Bhumiratana, A.; Flegel, T.W.; Glinsukon, T.; Shinmyo, A.

    1987-08-01

    A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.

  1. Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold.

    Science.gov (United States)

    Kalayanamitr, A; Bhumiratana, A; Flegel, T W; Glinsukon, T; Shinmyo, A

    1987-01-01

    A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production. PMID:2444160

  2. Sexual behavior of mutant strains of the medfly Ceratitis capitata (Diptera: Tephritidae

    Directory of Open Access Journals (Sweden)

    R.D Briceño

    2003-09-01

    Full Text Available Males of the mutant strains (blind, vestigal-winged of the Mediterranean fruit fly (medfly, Ceratits capitata (Wiedmann showed differences in behavior compared with control (mass-reared males. Mutant males made fewer mating attempts and achieved fewer matings than control males. Vestigal-winged females copulated less frequently with both mutants. Blind males climbed rather than jumped onto females and copulated in very low numbers compared with control and vestigal males. Blind females copulated normally with control, males and in very low numbers with both types of mutant malesMachos mutantes (ciegos, alas vestigiales de la mosca del mediterráneo Ceratitis capitata (Wiedmann mostraron diferencias en conducta comparados con los machos testigo (cría masiva. Los machos mutantes, realizaron menos intentos por aparearse y lograron menos apareamientos que los machos testigo. Las hembras con alas vestigiales, copularon menos con ambas clases de mutantes. Los machos ciegos, subieron en lugar de saltar sobre las hembras y copularon en números muy bajos comparados con los machos testigo y con los de alas vestigiales. Las hembras ciegas, copularon de forma normal con los machos testigo y en números muy bajos con ambos tipos de machos mutantes

  3. RECEPTOR POTENTIAL AND LIGHT-INDUCED MITOCHONDRIAL ACTIVATION IN BLOWFLY PHOTORECEPTOR MUTANTS

    NARCIS (Netherlands)

    MOJET, MH; TINBERGEN, J; STAVENGA, DG

    1991-01-01

    1. Simultaneous measurements of the receptor potential and the light-induced mitochondrial activation were performed in white-eyed blowflies Calliphora vicina, mutant chalky, and Lucilia cuprina, mutants w(F) and w'nss. The intensity dependence and the temporal dynamics were investigated. 2. The

  4. Elevated poly(3-hydroxybutyrate) synthesis in mutants of Ralstonia eutropha H16 defective in lipopolysaccharide biosynthesis

    NARCIS (Netherlands)

    Brandt, U.; Raberg, M.; Voigt, B.; Hecker, M.; Steinbuchel, A.

    2012-01-01

    Several independent transposon Tn5-induced mutants of Ralstonia eutropha H16 exhibited a poly(3-hydroxybutyric acid) (PHB) elevated phenotype and accumulated substantial amounts of PHB already in the exponential growth phase. The insertion loci of Tn5 in these six mutants were mapped in the genes

  5. Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta.

    Science.gov (United States)

    Jacobs, J. J.; Arroo, R. R.; De Koning, E. A.; Klunder, A. J.; Croes, A. F.; Wullems, G. J.

    1995-01-01

    Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta. These experiments also showed that one mutant is deficient in demethylation of the monothiophene. PMID:12228405

  6. Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants.

    Directory of Open Access Journals (Sweden)

    Özlem Demir

    2011-10-01

    Full Text Available The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants". Activity can be restored by second-site suppressor mutations ("rescue mutants". This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD, without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC metric was strongly correlated (r(2 = 0.77 with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i p53 cancer mutants were more flexible than wild-type protein, (ii second-site rescue mutations decreased the flexibility of cancer mutants, and (iii negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.

  7. Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

    DEFF Research Database (Denmark)

    Welin, M.; Skovgaard, T.; Knecht, Wolfgang

    2005-01-01

    The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3...

  8. “Start” Mutants of Saccharomyces cerevisiae Are Suppressed in Carbon Catabolite-Derepressing Medium

    OpenAIRE

    Shuster, Jeffrey R.

    1982-01-01

    Temperature-sensitive cell division “start” mutants cdc28, cdc36, cdc37, and cdc39 of the yeast Saccharomyces cerevisiae arrested cell division in the G1 phase of the cell cycle in glucose medium. I report here that cdc28, cdc36, and cdc39 mutants were suppressed when grown in carbon catabolite-derepressing medium.

  9. L-Glutamate production by lysozyme-sensitive Corynebacterium glutamicum ltsA mutant strains

    Directory of Open Access Journals (Sweden)

    Nagai Kazuo

    2001-10-01

    Full Text Available Abstract Background A non-pathogenic species of coryneform bacteria, Corynebacterium glutamicum, was originally isolated as an L-glutamate producing bacterium and is now used for fermentative production of various amino acids. A mutation in the C. glutamicum ltsA gene caused susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production. Results The characteristics of eight lysozyme-sensitive mutants which had been isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis were examined. Complementation analysis with the cloned wild-type ltsA gene and DNA sequencing of the ItsA region revealed that four mutants had a mutation in the ltsA gene. Among them, two mutants showed temperature-sensitive growth and overproduced L-glutamate at higher temperatures, as well as the previously reported ltsA mutant. Other two showed temperature-resistant growth: one missense mutant produced L-glutamate to some extent but the other nonsense mutant did not. These two mutants remained temperature-resistant in spite of introduction of ltsA::kan mutation that causes temperature-sensitive growth in the wild-type background. Conclusions These results indicate that a defect caused by the ltsA mutations is responsible for temperature-sensitive growth and L-glutamate overproduction by C. glutamicum. The two temperature-resistant mutants seem to carry suppressor mutations that rendered cells temperature-resistance and abolished L-glutamate overproduction.

  10. Induction, isolation, and characterization of aspergillus niger mutant strains producing elevated levels of beta-galactosidase.

    OpenAIRE

    Nevalainen, K M

    1981-01-01

    An Aspergillus niger mutant strain, VTT-D-80144, with an improvement of three- to fourfold in the production of extracellular beta-galactosidase was isolated after mutagenesis. The production of beta-galactosidase by this mutant was unaffected by fermentor size, and the enzyme was also suitable for immobilization.

  11. Induction, isolation, and characterization of aspergillus niger mutant strains producing elevated levels of beta-galactosidase.

    Science.gov (United States)

    Nevalainen, K M

    1981-01-01

    An Aspergillus niger mutant strain, VTT-D-80144, with an improvement of three- to fourfold in the production of extracellular beta-galactosidase was isolated after mutagenesis. The production of beta-galactosidase by this mutant was unaffected by fermentor size, and the enzyme was also suitable for immobilization. PMID:6784672

  12. Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence

    NARCIS (Netherlands)

    Jing, Hai-Chun; Sturre, Marcel J.G.; Hille, Jacques; Dijkwel, Paul P.

    2002-01-01

    The onset of leaf senescence is controlled by leaf age and ethylene can promote leaf senescence within a specific age window. We exploited the interaction between leaf age and ethylene and isolated mutants with altered leaf senescence that are named as onset of leaf death (old) mutants. Early leaf

  13. Photosynthetic Characteristics of Flag Leaves in Rice White Stripe Mutant 6001 During Senescence Process

    Directory of Open Access Journals (Sweden)

    Xiao-hui ZHEN

    2014-11-01

    Full Text Available Physiological, biochemical and electron microscopy analyses were used to investigate the photosynthetic performance of flag leaves in rice white stripe mutant 6001 during the senescence process. Results showed that the chlorophyll content at the heading and milk-ripe stages in rice mutant 6001 were about 34.78% and 3.00% less than those in wild type 6028, respectively. However, the chlorophyll content at the fully-ripe stage in rice mutant 6001 was higher than that in wild type 6028. At the heading stage, the net photosynthetic rate (Pn in rice mutant 6001 was lower than that in wild type 6028. Rice mutant 6001 also exhibited a significantly slower decrease rate of Pn than wild type 6028 during the senescence progress, especially at the later stage. Furthermore, Ca2+-ATPase, Mg2+-ATPase and photophosphorylation activities exhibited the similar trends as the Pn. During the senescence process, the 68 kDa polypeptide concentrations in the thylakoid membrane proteins exhibited a significant change, which was one of the critical factors that contributed to the observed change in photosynthesis. We also observed that the chloroplasts of rice mutant 6001 exhibited higher integrity than those of wild type 6028, and the chloroplast membrane of rice mutant 6001 disintegrated more slow during the senescence process. In general, rice mutant 6001 had a relatively slower senescence rate than wild type 6028, and exhibited anti-senescence properties.

  14. Molecular mapping of three nuclear male sterility mutant genes in cultivated sunflower (Helianthus annuus L.)

    Science.gov (United States)

    The nuclear male sterility (NMS) trait is a useful tool for sunflower (Helianthus annuus L.) breeding and genetic programs. Previously, we induced NMS mutants in cultivated line HA 89. The mutants possessed single recessive genes, ms6, ms7, and ms8, respectively, in NMS HA 89-872, NMS HA 89-552, and...

  15. Chemical Excitation and Inactivation in Photoreceptors of the Fly Mutants trp and nss

    NARCIS (Netherlands)

    Suss, E.; Barash, S.; Stavenga, D.G.; Stieve, H.; Selinger, Z.; Minke, B.

    1989-01-01

    The Drosophila and Lucilia photoreceptor mutants, trp and nss, respond like wild-type flies to a short pulse of intense light or prolonged dim light; however, upon continuous intense illumination, the trp and nss mutants are unable to maintain persistent excitation. This defect manifests itself by a

  16. Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha : A physiological study

    NARCIS (Netherlands)

    Klei, Ida J. van der; Harder, Willem; Veenhuis, Marten

    We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wildtype (WT) levels, this mutant is unable to grow

  17. Comparative production of cellulases by mutants of Trichoderma parceramosume PTCC5140

    Directory of Open Access Journals (Sweden)

    Hoda Nouri

    2017-06-01

    Discussion and conclusion: Evaluation of cellulase production in mutant strains of Trichoderma parceramosume PTCC 5140 showed that use of chemical mutagenesis with 2 to 11 fold increasing in enzyme activity is a potent method to improve cellulase complex activity. In the current study, obtained mutant strains could be introduced as a potent cellulase producer for further studies in bioconversion processes.

  18. Rescue of ligand binding of a mutant IGF-I receptor by complementation

    DEFF Research Database (Denmark)

    Chakravarty, Arjun Anders; Hinrichsen, Jane; Whittaker, Linda

    2005-01-01

    from a wild-type receptor monomer and a mutant receptor monomer devoid of binding activity. Receptor hybrids were generated by transient co-transfection of cDNAs encoding wild-type and mutant receptors with unique epitope tags. Hybrid receptors were purified from transfected cells by sequential immuno...

  19. Leaf Rolling and Stem Fasciation in Grass Pea (Lathyrus sativus L. Mutant Are Mediated through Glutathione-Dependent Cellular and Metabolic Changes and Associated with a Metabolic Diversion through Cysteine during Phenotypic Reversal

    Directory of Open Access Journals (Sweden)

    Dibyendu Talukdar

    2014-01-01

    Full Text Available A Lathyrus sativus L. mutant isolated in ethylmethane sulfonate-treated M2 progeny of mother variety BioL-212 and designated as rlfL-1 was characterized by inwardly rolled-leaf and stem and bud fasciations. The mutant exhibited karyomorphological peculiarities in both mitosis and meiosis with origin of aneuploidy. The mitosis was vigorous with high frequency of divisional cells and their quick turnover presumably steered cell proliferations. Significant transcriptional upregulations of cysteine and glutathione synthesis and concomitant stimulations of glutathione-mediated antioxidant defense helped rlfL-1 mutant to maintain balanced reactive oxygen species (ROS metabolisms, as deduced by ROS-imaging study. Glutathione synthesis was shut down in buthionine sulfoximine- (BSO- treated mother plant and mutant, and leaf-rolling and stems/buds fasciations in the mutant were reversed, accompanied by normalization of mitotic cell division process. Antioxidant defense was downregulated under low glutathione-redox but cysteine-desulfurations and photorespiratory glycolate oxidase transcripts were markedly overexpressed, preventing cysteine overaccumulation but resulted in excess H2O2 in BSO-treated mutant. This led to oxidative damage in proliferating cells, manifested by severe necrosis in rolled-leaf and fasciated stems. Results indicated vital role of glutathione in maintaining abnormal proliferations in plant organs, and its deficiency triggered phenotypic reversal through metabolic diversions of cysteine and concomitant cellular and metabolic modulations.

  20. phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens

    OpenAIRE

    Elsheimer-Matulova, Marta; Varmuzova, Karolina; Kyrova, Kamila; Havlickova, Hana; Sisak, Frantisek; Rahman, Masudur; Rychlik, Ivan

    2015-01-01

    Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitorin...

  1. Guanylate cyclase 2C agonism corrects CFTR mutants.

    Science.gov (United States)

    Arora, Kavisha; Huang, Yunjie; Mun, Kyushik; Yarlagadda, Sunitha; Sundaram, Nambirajan; Kessler, Marco M; Hannig, Gerhard; Kurtz, Caroline B; Silos-Santiago, Inmaculada; Helmrath, Michael; Palermo, Joseph J; Clancy, John P; Steinbrecher, Kris A; Naren, Anjaparavanda P

    2017-10-05

    Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate-synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient-derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.

  2. Bacteriophage-insensitive mutants for high quality Crescenza manufacture

    Directory of Open Access Journals (Sweden)

    Donatella eChirico

    2014-05-01

    Full Text Available Streptococcus thermophilus is a thermophilic lactic acid bacterium used as starter culture for the manufacture of fermented dairy products. For the production of Crescenza and other soft cheeses, Sacco has developed and provides dairies with 3 different defined blends of S. thermophilus strains. Each blend contains 2 different S. thermophilus strains. The strains were selected based on their unique technological properties as well as different phage profiles. Analysis of 133 whey samples collected in 2009-2010 from Italian dairies showed a high prevalence (about 50% of bacteriophage attacks on the blend ST020. More specifically, the strain S. thermophilus ST1A was found to be the preferred target of the bacteriophages. A bacteriophage insensitive mutant (BIM5 of the phage-sensitive strain ST1A was successfully developed and used to substitute strain ST1A in the Crescenza starter culture ST020. The strain BIM5 showed identical technological and industrial traits as those of the phage-sensitive strain ST1A. The improved resistance of the modified Crescenza starter culture ST020R was confirmed at Italian dairies, and its effectiveness monitored on 122 whey samples collected in 2011-2012. Compared to the previous values (2009-2010, the use of the phage-hardened blend ST020R allowed reducing of frequency of phage attacks from about 50 to less than 5% of the whey samples investigated.

  3. Fecal corticosterone levels in RCAN1 mutant mice.

    Science.gov (United States)

    Rakowski-Anderson, Tammy; Wong, Helen; Rothermel, Beverly; Cain, Peter; Lavilla, Carmencita; Pullium, Jennifer K; Hoeffer, Charles

    2012-04-01

    Regulator of calcineurin 1 (RCAN1) is related to the expression of human neurologic disorders such as Down syndrome, Alzheimer disease, and chromosome 21q deletion syndrome. We showed here that RCAN1-knockout mice exhibit reduced innate anxiety as indicated by the elevated-plus maze. To examine whether glucocorticoids contribute to this phenotype, we measured fecal corticosterone in male wildtype and RCAN1-knockout mice and in male and female transgenic mice with neuronal overexpression of RCAN1 (Tg-RCAN1(TG)). We found no difference in fecal corticosterone levels of RCAN1-knockout mice and their wildtype littermates. As expected, we found differences between sexes in fecal corticosterone levels. In addition, we found higher levels of excreted corticosterone in Tg-RCAN1(TG) female mice as compared with female wildtype mice. Our data indicate normal diurnal corticosterone production in RCAN1 mutant mice and do not suggest a causal role in either the cognitive or anxiety phenotypes exhibited by RCAN1-knockout mice.

  4. Proteomic and Genomic Analyses of Antimony Resistant Leishmania infantum Mutant

    Science.gov (United States)

    Brotherton, Marie-Christine; Bourassa, Sylvie; Leprohon, Philippe; Légaré, Danielle; Poirier, Guy G.; Droit, Arnaud; Ouellette, Marc

    2013-01-01

    Background Antimonials remain the primary antileishmanial drugs in most developing countries. However, drug resistance to these compounds is increasing and our understanding of resistance mechanisms is partial. Methods/Principal Findings In the present study, quantitative proteomics using stable isotope labelling of amino acids in cell culture (SILAC) and genome next generation sequencing were used in order to better characterize in vitro generated Leishmania infantum antimony resistant mutant (Sb2000.1). Using the proteomic method, 58 proteins were found to be differentially regulated in Sb2000.1. The ABC transporter MRPA (ABCC3), a known marker of antimony resistance, was observed for the first time in a proteomic screen. Furthermore, transfection of its gene conferred antimony resistance in wild-type cells. Next generation sequencing revealed aneuploidy for 8 chromosomes in Sb2000.1. Moreover, specific amplified regions derived from chromosomes 17 and 23 were observed in Sb2000.1 and a single nucleotide polymorphism (SNP) was detected in a protein kinase (LinJ.33.1810-E629K). Conclusion/Significance Our results suggest that differentially expressed proteins, chromosome number variations (CNVs), specific gene amplification and SNPs are important features of antimony resistance in Leishmania. PMID:24312377

  5. Bacteriophage-insensitive mutants for high quality Crescenza manufacture.

    Science.gov (United States)

    Chirico, Donatella; Gorla, Arianna; Verga, Viola; Pedersen, Per D; Polgatti, Eliseo; Cava, Antonio; Dal Bello, Fabio

    2014-01-01

    Streptococcus thermophilus is a thermophilic lactic acid bacterium used as starter culture for the manufacture of fermented dairy products. For the production of Crescenza and other soft cheeses, Sacco has developed and provides dairies with three different defined blends of S. thermophilus strains. Each blend contains two different S. thermophilus strains. The strains were selected based on their unique technological properties as well as different phage profiles. Analysis of 133 whey samples collected in 2009-2010 from Italian dairies showed a high prevalence (about 50%) of bacteriophage attacks on the blend ST020. More specifically, the strain S. thermophilus ST1A was found to be the preferred target of the bacteriophages. A bacteriophage insensitive mutant (BIM5) of the phage-sensitive strain ST1A was successfully developed and used to substitute strain ST1A in the Crescenza starter culture ST020. The strain BIM5 showed identical technological and industrial traits as those of the phage-sensitive strain ST1A. The improved resistance of the modified Crescenza starter culture ST020R was confirmed at Italian dairies, and its effectiveness monitored on 122 whey samples collected in 2011-2012. Compared to the previous values (2009-2010), the use of the phage-hardened blend ST020R allowed reducing of frequency of phage attacks from about 50 to less than 5% of the whey samples investigated.

  6. Producing Conditional Mutants for Studying Plant Microtubule Function

    Energy Technology Data Exchange (ETDEWEB)

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  7. WEDGE: An anticoagulant thrombin mutant produced by autoactivation

    Science.gov (United States)

    Wood, D.C.; Pelc, L.A.; Pozzi, N.; Wallisch, M.; Verbout, N.G.; Tucker, E.I.; Gruber, A.; Di Cera, E.

    2015-01-01

    Summary Background Production of therapeutically relevant proteases typically involves activation of a zymogen precursor by external enzymes, which may raise regulatory issues about availability and purity. Recent studies of thrombin precursors have shown how to engineer constructs that spontaneously convert to the mature protease by autoactivation, without the need of external enzymes. Objectives Autoactivation is an innovative strategy that promises to simplify the production of proteases of therapeutic relevance, but has not been tested in practical applications. This study aims to provide a direct test of this strategy. Methods An autoactivating version of the thrombin mutant W215A/E217A (WE), currently in pre-clinical development as an anticoagulant, is engineered. Results and Conclusions The autoactivating version of WE can be produced in large quantities, like WE made in BHK cells or E. coli, and retains all significant functional properties in vitro and in vivo. The results serve as proof of principle that autoactivation is an innovative and effective strategy for production of trypsin-like proteases of therapeutic relevance. PMID:25369995

  8. Isolating human DNA repair genes using rodent-cell mutants

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  9. Mutant WD-repeat protein in triple-A syndrome.

    Science.gov (United States)

    Tullio-Pelet, A; Salomon, R; Hadj-Rabia, S; Mugnier, C; de Laet, M H; Chaouachi, B; Bakiri, F; Brottier, P; Cattolico, L; Penet, C; Bégeot, M; Naville, D; Nicolino, M; Chaussain, J L; Weissenbach, J; Munnich, A; Lyonnet, S

    2000-11-01

    Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Whereas several lines of evidence indicate that triple-A syndrome results from the abnormal development of the autonomic nervous system, late-onset progressive neurological symptoms (including cerebellar ataxia, peripheral neuropathy and mild dementia) suggest that the central nervous system may be involved in the disease as well. Using fine-mapping based on linkage disequilibrium in North African inbred families, we identified a short ancestral haplotype on chromosome 12q13 (<1 cM), sequenced a BAC contig encompassing the triple-A minimal region and identified a novel gene (AAAS) encoding a protein of 547 amino acids that is mutant in affected individuals. We found five homozygous truncating mutations in unrelated patients and ascribed the founder effect in North African families to a single splice-donor site mutation that occurred more than 2,400 years ago. The predicted product of AAAS, ALADIN (for alacrima-achalasia-adrenal insufficiency neurologic disorder), belongs to the WD-repeat family of regulatory proteins, indicating a new disease mechanism involved in triple-A syndrome. The expression of the gene in both neuroendocrine and cerebral structures points to a role in the normal development of the peripheral and central nervous systems.

  10. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  11. Natural variation of model mutant phenotypes in Ciona intestinalis.

    Directory of Open Access Journals (Sweden)

    Paolo Sordino

    Full Text Available BACKGROUND: The study of ascidians (Chordata, Tunicata has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. CONCLUSIONS/SIGNIFICANCE: Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.

  12. Demarcation of mutant-carrying regions in barley plants after ethylmethane-sulfonate seed treatment

    DEFF Research Database (Denmark)

    Jacobsen, P.

    1966-01-01

    The branching pattern of the barley plant is analyzed and the anatomical structure of the resting barley embryo studied in longitudinal and cross-sections as well as by dissection techniques. The frequency and distribution of ethylmethane-sulfonate induced chloroplast and morphological seedling...... mutants were analyzed in spikes classified according to their ontogenetic relationship. The frequency with which two spikes segregated identical mutants was determined by pairwise comparisons of all spikes in each plant. In this way the frequency of mutant cluster sharing between spikes and spike groups...... was obtained.The absence of cluster sharing allows the recognition in the barley plant of 8 mutually exclusive mutant sectors which never had a mutant cluster in common. The anatomical analysis proves that the barley embryo contains at least 6 separate shoot meristems or prospective shoot meristems, which...

  13. Isolation and characterization of a barley mutant with abscisic-acid-insensitive stomata.

    Science.gov (United States)

    Raskin, I; Ladyman, J A

    1988-01-01

    A barley (Hordeum vulgare L.) mutant ("cool") with leaf transpiration unaffected by the application of 1 mM abscisic acid (ABA) was isolated from the population of M2 seedlings using thermography (electronic visualization, and quantitation of the temperature profiles on the surface of the leaves). Stomata of the mutant plants were insensitive to exogenously applied ABA, darkness, and such desiccation treatments as leaf excision and drought stress. The evaporative cooling of the leaves of the "cool" barley was always higher than that of the wild-type barley, even without ABA application, indicating that the diffusive resistance of the mutant leaves to water loss was always lower. Guard-cell morphology and stomatal density as well as ABA level and metabolism were seemingly unaltered in the mutant plants. In addition, gibberellin-induced α-amylase secretion and precocious embryo germination in the mutant barley was inhibited by ABA to the same extent as in the wild-type barley.

  14. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

    Science.gov (United States)

    Amorim-Vaz, Sara; Delarze, Eric; Ischer, Françoise; Sanglard, Dominique; Coste, Alix T

    2015-01-01

    The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

  15. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models

    Directory of Open Access Journals (Sweden)

    Sara eAmorim-Vaz

    2015-05-01

    Full Text Available The aim of the present study was to identify C. albicans transcription factors (TF involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens quantified in kidneys. Mutants of unannotated genes which generated a kidney fungal burden significantly different from that of wild-type were selected and individually examined in G. mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25 % of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects, a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching fungal burden phenotypes were observed in 50 % of the cases, highlighting the bias due to host effects. In contrast, 33.4 % concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the pool effect. After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adaptation.

  16. Mutant p53 upregulates alpha-1 antitrypsin expression and promotes invasion in lung cancer.

    Science.gov (United States)

    Shakya, R; Tarulli, G A; Sheng, L; Lokman, N A; Ricciardelli, C; Pishas, K I; Selinger, C I; Kohonen-Corish, M R J; Cooper, W A; Turner, A G; Neilsen, P M; Callen, D F

    2017-08-01

    Missense mutations in the TP53 tumor-suppressor gene inactivate its antitumorigenic properties and endow the incipient cells with newly acquired oncogenic properties that drive invasion and metastasis. Although the oncogenic effect of mutant p53 transcriptome has been widely acknowledged, the global influence of mutant p53 on cancer cell proteome remains to be fully elucidated. Here, we show that mutant p53 drives the release of invasive extracellular factors (the 'secretome') that facilitates the invasion of lung cancer cell lines. Proteomic characterization of the secretome from mutant p53-inducible H1299 human non-small cell lung cancer cell line discovered that the mutant p53 drives its oncogenic pathways through modulating the gene expression of numerous targets that are subsequently secreted from the cells. Of these genes, alpha-1 antitrypsin (A1AT) was identified as a critical effector of mutant p53 that drives invasion in vitro and in vivo, together with induction of epithelial-mesenchymal transition markers expression. Mutant p53 upregulated A1AT transcriptionally through the involvement with its family member p63. Conditioned medium containing secreted A1AT enhanced cell invasion, while an A1AT-blocking antibody attenuated the mutant p53-driven migration and invasion. Importantly, high A1AT expression correlated with increased tumor stage, elevated p53 staining and shorter overall survival in lung adenocarcinoma patients. Collectively, these findings suggest that A1AT is an indispensable target of mutant p53 with prognostic and therapeutic potential in mutant p53-expressing tumors.

  17. Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Tani, Tatsunori; Taguchi, Hisataka; Fujimori, Kazuhiro E; Sahara, Takehiko; Ohgiya, Satoru; Kamagata, Yoichi; Akamatsu, Takashi

    2016-10-01

    To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    Science.gov (United States)

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

  19. Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism.

    Science.gov (United States)

    Cushman, John C; Agarie, Sakae; Albion, Rebecca L; Elliot, Stewart M; Taybi, Tahar; Borland, Anne M

    2008-05-01

    Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

  20. Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

    Science.gov (United States)

    Pusch, Stefan; Krausert, Sonja; Fischer, Viktoria; Balss, Jörg; Ott, Martina; Schrimpf, Daniel; Capper, David; Sahm, Felix; Eisel, Jessica; Beck, Ann-Christin; Jugold, Manfred; Eichwald, Viktoria; Kaulfuss, Stefan; Panknin, Olaf; Rehwinkel, Hartmut; Zimmermann, Katja; Hillig, Roman C; Guenther, Judith; Toschi, Luisella; Neuhaus, Roland; Haegebart, Andrea; Hess-Stumpp, Holger; Bauser, Markus; Wick, Wolfgang; Unterberg, Andreas; Herold-Mende, Christel; Platten, Michael; von Deimling, Andreas

    2017-04-01

    Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

  1. Biological phosphorylation of an Unnatural Base Pair (UBP using a Drosophila melanogaster deoxynucleoside kinase (DmdNK mutant.

    Directory of Open Access Journals (Sweden)

    Fei Chen

    Full Text Available One research goal for unnatural base pair (UBP is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK, which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1'-b-d-2'-deoxyribofuranosyl-2(1H-pyridone, Z and its complementary purine analogue (2-amino-8-(1'-b-d-2'-deoxyribofuranosyl-imidazo[1,2-a]-1,3,5-triazin-4(8H-one, P. The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo.

  2. Pseudomonas fluorescens F113 Mutant with Enhanced Competitive Colonization Ability and Improved Biocontrol Activity against Fungal Root Pathogens ▿

    Science.gov (United States)

    Barahona, Emma; Navazo, Ana; Martínez-Granero, Francisco; Zea-Bonilla, Teresa; Pérez-Jiménez, Rosa María; Martín, Marta; Rivilla, Rafael

    2011-01-01

    Motility is one of the most important traits for efficient rhizosphere colonization by Pseudomonas fluorescens F113rif (F113). In this bacterium, motility is a polygenic trait that is repressed by at least three independent pathways, including the Gac posttranscriptional system, the Wsp chemotaxis-like pathway, and the SadB pathway. Here we show that the kinB gene, which encodes a signal transduction protein that together with AlgB has been implicated in alginate production, participates in swimming motility repression through the Gac pathway, acting downstream of the GacAS two-component system. Gac mutants are impaired in secondary metabolite production and are unsuitable as biocontrol agents. However, the kinB mutant and a triple mutant affected in kinB, sadB, and wspR (KSW) possess a wild-type phenotype for secondary metabolism. The KSW strain is hypermotile and more competitive for rhizosphere colonization than the wild-type strain. We have compared the biocontrol activity of KSW with those of the wild-type strain and a phenotypic variant (F113v35 [V35]) which is hypermotile and hypercompetitive but is affected in secondary metabolism since it harbors a gacS mutation. Biocontrol experiments in the Fusarium oxysporum f. sp. radicis-lycopersici/Lycopersicum esculentum (tomato) and Phytophthora cactorum/Fragaria vesca (strawberry) pathosystems have shown that the three strains possess biocontrol activity. Biocontrol activity was consistently lower for V35, indicating that the production of secondary metabolites was the most important trait for biocontrol. Strain KSW showed improved biocontrol compared with the wild-type strain, indicating that an increase in competitive colonization ability resulted in improved biocontrol and that the rational design of biocontrol agents by mutation is feasible. PMID:21685161

  3. Pseudomonas fluorescens F113 mutant with enhanced competitive colonization ability and improved biocontrol activity against fungal root pathogens.

    Science.gov (United States)

    Barahona, Emma; Navazo, Ana; Martínez-Granero, Francisco; Zea-Bonilla, Teresa; Pérez-Jiménez, Rosa María; Martín, Marta; Rivilla, Rafael

    2011-08-01

    Motility is one of the most important traits for efficient rhizosphere colonization by Pseudomonas fluorescens F113rif (F113). In this bacterium, motility is a polygenic trait that is repressed by at least three independent pathways, including the Gac posttranscriptional system, the Wsp chemotaxis-like pathway, and the SadB pathway. Here we show that the kinB gene, which encodes a signal transduction protein that together with AlgB has been implicated in alginate production, participates in swimming motility repression through the Gac pathway, acting downstream of the GacAS two-component system. Gac mutants are impaired in secondary metabolite production and are unsuitable as biocontrol agents. However, the kinB mutant and a triple mutant affected in kinB, sadB, and wspR (KSW) possess a wild-type phenotype for secondary metabolism. The KSW strain is hypermotile and more competitive for rhizosphere colonization than the wild-type strain. We have compared the biocontrol activity of KSW with those of the wild-type strain and a phenotypic variant (F113v35 [V35]) which is hypermotile and hypercompetitive but is affected in secondary metabolism since it harbors a gacS mutation. Biocontrol experiments in the Fusarium oxysporum f. sp. radicis-lycopersici/Lycopersicum esculentum (tomato) and Phytophthora cactorum/Fragaria vesca (strawberry) pathosystems have shown that the three strains possess biocontrol activity. Biocontrol activity was consistently lower for V35, indicating that the production of secondary metabolites was the most important trait for biocontrol. Strain KSW showed improved biocontrol compared with the wild-type strain, indicating that an increase in competitive colonization ability resulted in improved biocontrol and that the rational design of biocontrol agents by mutation is feasible.

  4. BRAF Fusion as a Novel Mechanism of Acquired Resistance to Vemurafenib in BRAFV600E Mutant Melanoma.

    Science.gov (United States)

    Kulkarni, Atul; Al-Hraishawi, Husam; Simhadri, Srilatha; Hirshfield, Kim M; Chen, Suzie; Pine, Sharon; Jeyamohan, Chandrika; Sokol, Levi; Ali, Siraj; Teo, Man Lung; White, Eileen; Rodriguez-Rodriguez, Lorna; Mehnert, Janice M; Ganesan, Shridar

    2017-09-15

    Purpose: Many patients with BRAFV600E mutant melanoma treated with BRAF inhibitors experience a rapid response, but ultimately develop resistance. Insight into the mechanism of resistance is critical for development of more effective treatment strategies.Experimental Design: Comprehensive genomic profiling of serial biopsies was performed in a patient with a BRAFV600E mutant metastatic melanoma who developed resistance to vemurafenib. An AGAP3-BRAF fusion gene, identified in the vemurafenib-resistant tumor, was expressed in BRAFV600E melanoma cell lines, and its effect on drug sensitivity was evaluated.Results: Clinical resistance to vemurafenib in a melanoma harboring a BRAFV600E mutation was associated with acquisition of an AGAP3-BRAF fusion gene. Expression of the AGAP3-BRAF fusion in BRAFV600E mutant melanoma cells induced vemurafenib resistance; however, these cells remained relatively sensitive to MEK inhibitors. The patient experienced clinical benefit following treatment with the combination of a BRAF and a MEK inhibitor. Rebiopsy of the tumor at a later time point, after BRAF and MEK inhibitors had been discontinued, showed loss of the AGAP3-BRAF fusion gene. Mixing experiments suggest that cells harboring both BRAFV600E and AGAP3-BRAF only have a fitness advantage over parental BRAFV600E cells during active treatment with a BRAF inhibitor.Conclusions: We report acquisition of a BRAF fusion as a novel mechanism of acquired resistance to vemurafenib in a patient with melanoma harboring a BRAFV600E mutation. The acquisition and regression of clones harboring this fusion during the presence and absence of a BRAF inhibitor are consistent with rapidly evolving clonal dynamics in melanoma. Clin Cancer Res; 23(18); 5631-8. ©2017 AACR. ©2017 American Association for Cancer Research.

  5. Drosophila Interspecific Hybrids Phenocopy piRNA-Pathway Mutants

    Science.gov (United States)

    Kelleher, Erin S.; Edelman, Nathaniel B.; Barbash, Daniel A.

    2012-01-01

    The Piwi-interacting RNA (piRNA) pathway defends the germline of animals from the deleterious activity of selfish transposable elements (TEs) through small-RNA mediated silencing. Adaptation to novel invasive TEs is proposed to occur by incorporating their sequences into the piRNA pool that females produce and deposit into their eggs, which then propagates immunity against specific TEs to future generations. In support of this model, the F1 offspring of crosses between strains of the same Drosophila species sometimes suffer from germline derepression of paternally inherited TE families, caused by a failure of the maternal strain to produce the piRNAs necessary for their regulation. However, many protein components of the Drosophila piRNA pathway exhibit signatures of positive selection, suggesting that they also contribute to the evolution of host genome defense. Here we investigate piRNA pathway function and TE regulation in the F1 hybrids of interspecific crosses between D. melanogaster and D. simulans and compare them with intraspecific control crosses of D. melanogaster. We confirm previous reports showing that intraspecific crosses are characterized by derepression of paternally inherited TE families that are rare or absent from the maternal genome and piRNA pool, consistent with the role of maternally deposited piRNAs in shaping TE silencing. In contrast to the intraspecific cross, we discover that interspecific hybrids are characterized by widespread derepression of both maternally and paternally inherited TE families. Furthermore, the pattern of derepression of TE families in interspecific hybrids cannot be attributed to their paucity or absence from the piRNA pool of the maternal species. Rather, we demonstrate that interspecific hybrids closely resemble piRNA effector-protein mutants in both TE misregulation and aberrant piRNA production. We suggest that TE derepression in interspecific hybrids largely reflects adaptive divergence of piRNA pathway genes

  6. Catalytic properties of thimet oligopeptidase H600A mutant

    Energy Technology Data Exchange (ETDEWEB)

    Machado, Mauricio F.M.; Marcondes, Marcelo F. [Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil); Rioli, Vanessa [Laboratorio Especial de Toxinologia Aplicada, Instituto Butantan, 05467-010 Sao Paulo, SP (Brazil); Departamento de Biologia Celular e Desenvolvimento, Universidade de Sao Paulo, 05508-900 Sao Paulo, SP (Brazil); Ferro, Emer S. [Departamento de Biologia Celular e Desenvolvimento, Universidade de Sao Paulo, 05508-900 Sao Paulo, SP (Brazil); Juliano, Maria A.; Juliano, Luiz [Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil); Oliveira, Vitor, E-mail: vitor.oliveira@unifesp.br [Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil)

    2010-04-02

    Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His{sup 600}. In the present work, the role of His{sup 600} of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S{sub 1} and S{sub 1}' specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His{sup 600} residue makes important interactions with the substrate, supporting the prediction that His{sup 600} moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K{sub m} and k{sub cat}, showing the importance of His{sup 600} for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His{sup 600} in TOP catalysis, transferring a proton to the newly generated NH{sub 2}-terminus or helping Tyr{sup 605} and/or Tyr{sup 612} in the intermediate oxyanion stabilization.

  7. Genetics of peripheral vestibular dysfunction: lessons from mutant mouse strains.

    Science.gov (United States)

    Jones, Sherri M; Jones, Timothy A

    2014-03-01

    A considerable amount of research has been published about genetic hearing impairment. Fifty to sixty percent of hearing loss is thought to have a genetic cause. Genes may also play a significant role in acquired hearing loss due to aging, noise exposure, or ototoxic medications. Between 1995 and 2012, over 100 causative genes have been identified for syndromic and nonsyndromic forms of hereditary hearing loss. Mouse models have been extremely valuable in facilitating the discovery of hearing loss genes and in understanding inner ear pathology due to genetic mutations or elucidating fundamental mechanisms of inner ear development. Whereas much is being learned about hereditary hearing loss and the genetics of cochlear disorders, relatively little is known about the role genes may play in peripheral vestibular impairment. Here we review the literature with regard to genetics of vestibular dysfunction and discuss what we have learned from studies using mutant mouse models and direct measures of peripheral vestibular neural function. Several genes are considered that when mutated lead to varying degrees of inner ear vestibular dysfunction due to deficits in otoconia, stereocilia, hair cells, or neurons. Behavior often does not reveal the inner ear deficit. Many of the examples presented are also known to cause human disorders. Knowledge regarding the roles of particular genes in the operation of the vestibular sensory apparatus is growing, and it is clear that gene products co-expressed in the cochlea and vestibule may play different roles in the respective end organs. The discovery of new genes mediating critical inner ear vestibular function carries the promise of new strategies in diagnosing, treating, and managing patients as well as predicting the course and level of morbidity in human vestibular disease. American Academy of Audiology.

  8. Distinctive Klf4 mutants determine preference for DNA methylation status

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Hideharu; Wang, Dongxue; Steves, Alyse N.; Jin, Peng; Blumenthal, Robert M.; Zhang, Xing; Cheng, Xiaodong

    2016-09-04

    Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.

  9. Prompting Designers to Design

    DEFF Research Database (Denmark)

    Ahmed, Saeema

    2006-01-01

    Recent research suggest that engineering designers need assistance to understand what information is relevant for their particular design problem. They require guidance in formulating their queries and also to understand what information is relevant for them. This paper presents an approach...... to prompt designers with their design queries. A method that automatically extracts relationships between concepts is described, along with some examples. The method can be implemented as part of knowledge management system and the relationships are extracted form documents that are indexed within...... the system. The distinctive features of this approach is that all the concepts are elicited from the minds of engineering designers, and the system builds up knowledge as more documents enter the system. The approach is based on an understanding obtained from a number of empirical studies and also from...

  10. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2015-08-01

    Full Text Available Apolipoprotein C-II (APOC2 is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

  11. Effect of Mutant p53 Proteins on Glycolysis and Mitochondrial Metabolism.

    Science.gov (United States)

    Eriksson, Matilda; Ambroise, Gorbatchev; Ouchida, Amanda Tomie; Lima Queiroz, Andre; Smith, Dominique; Gimenez-Cassina, Alfredo; Iwanicki, Marcin P; Muller, Patricia A; Norberg, Erik; Vakifahmetoglu-Norberg, Helin

    2017-12-15

    TP53 is one of the most commonly mutated genes in human cancers. Unlike other tumor suppressors that are frequently deleted or acquire loss-of-function mutations, the majority of TP53 mutations in tumors are missense substitutions, which lead to the expression of full-length mutant proteins that accumulate in cancer cells and may confer unique gain-of-function (GOF) activities to promote tumorigenic events. Recently, mutant p53 proteins have been shown to mediate metabolic changes as a novel GOF to promote tumor development. There is a strong rationale that the GOF activities, including alterations in cellular metabolism, might vary between the different p53 mutants. Accordingly, the effect of different mutant p53 proteins on cancer cell metabolism is largely unknown. In this study, we have metabolically profiled several individual frequently occurring p53 mutants in cancers, focusing on glycolytic and mitochondrial oxidative phosphorylation pathways. Our investigation highlights the diversity of different p53 mutants in terms of their effect on metabolism, which might provide a foundation for the development of more effective targeted pharmacological approaches toward variants of mutant p53. Copyright © 2017 American Society for Microbiology.

  12. Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems.

    Science.gov (United States)

    Gao, L J; Jia, Y L; Li, S K; Qiu, L L

    2015-10-27

    The aim of the present study was to isolate and characterize novel nitrate reductase (NR)-deficient mutants, which may be useful for the transgenic manipulation of Dunaliella salina. Three NR-deficient mutants of D. salina, J-1, J-2, and J-3, were successfully isolated by screening for chlorate resistance after chemical mutagenesis with ethylnitrosourea. NR activity was not detected in the mutants and the expression of NR mRNA was significantly decreased. Growth analysis of D. salina strains grown in media containing different nitrogen sources revealed that these mutants were capable of utilizing nitrite and urea, but not nitrate as a nitrogen source, indicating that these mutants are indeed NR-deficient. Mutation analysis of NR cDNA sequences revealed that there were 11 point mutations shared by the J-1, J-2, and J-3 mutants. Furthermore, the results of the functional complementation experiment showed that NR activity of transformant T-1 derived from J-1 was recovered to 48.1 % of that of the wild-type D. salina. The findings of the present study indicate that nitrate may be used as a selective agent rather than antibiotics or herbicides for the isolated NR-deficient mutants in future transgenic D. salina systems.

  13. Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA.

    Science.gov (United States)

    Malena, Adriana; Pantic, Boris; Borgia, Doriana; Sgarbi, Gianluca; Solaini, Giancarlo; Holt, Ian J; Spinazzola, Antonella; Perissinotto, Egle; Sandri, Marco; Baracca, Alessandra; Vergani, Lodovica

    2016-11-01

    Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.

  14. TP53 mutants in the tower of babel of cancer progression.

    Science.gov (United States)

    Bisio, Alessandra; Ciribilli, Yari; Fronza, Gilberto; Inga, Alberto; Monti, Paola

    2014-06-01

    Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive, interfering, contact, structural, unfolded, misfolded, dimeric, monomeric, non-cooperative, unstable, supertrans, superstable, intragenic suppressor. TP53 mutants are many, more than 2,000 in fact, and they can be very diverse. Sporadic; germline; gain-of-function (GoF); oncogenic; rebel-angel; yin and yang; prion-like; metastasis-inducer; mediator of chemo-resistance; modifier of stemness. TP53 mutants can impact important cancer clinical variables, in multiple, often subtle ways, as revealed by cell-based assays as well as animal models. Here, we review studies investigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recent findings on how TP53 mutants can exhibit GoF properties. We also review reports on TP53 mutants' impact on cancer cell transcriptomes and studies with Li-Fraumeni patients trying to classify and predict phenotypes in relation to experimentally determined transcription fingerprints. Finally, we provide an example of the complexity of correlating TP53 mutant functionality to clinical variables in sporadic cancer patients. Conflicting results and limitations of experimental approaches notwithstanding, the study of TP53 mutants has provided a rich body of knowledge, mostly available in the public domain and accessible through databases, which is beginning to impact cancer intervention strategies. © 2014 WILEY PERIODICALS, INC.

  15. Production and Characterization of Radiation-Sensitive Meiotic Mutants of Coprinus Cinereus

    Science.gov (United States)

    Zolan, M. E.; Tremel, C. J.; Pukkila, P. J.

    1988-01-01

    We have isolated four γ-ray-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1;rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the few viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants. PMID:3197952

  16. Recovery of the wild type atomic flexibility in the HIV-1 protease double mutants.

    Science.gov (United States)

    De Conto, Valderes; Braz, Antônio S K; Perahia, David; Scott, Luis P B

    2015-06-01

    The emergence of drug resistant mutations due to the selective pressure exerted by antiretrovirals, including protease inhibitors (PIs), remains a major problem in the treatment of AIDS. During PIs therapy, the occurrence of primary mutations in the wild type HIV-1 protease reduces both the affinity for the inhibitors and the viral replicative capacity compared to the wild type (WT) protein, but additional mutations compensate for this reduced viral fitness. To investigate this phenomenon from the structural point of view, we combined Molecular Dynamics and Normal Mode Analysis to analyze and compare the variations of the flexibility of C-alpha atoms and the differences in hydrogen bond (h-bond) network between the WT and double mutants. In most cases, the flexibility profile of the double mutants was more often similar to that of the WT than to that of the related single base mutants. All single mutants showed a significant alteration in h-bond formation compared to WT. Most of the significant changes occur in the border between the flap and cantilever regions. We found that all the considered double mutants have their h-bond pattern significantly altered in comparison to the respective single base mutants affecting their flexibility profile that becomes more similar to that of WT. This WT flexibility restoration in the double mutants appears as an important factor for the HIV-1 fitness recovery observed in patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Atrial fibrillation-linked germline GJA5/connexin40 mutants showed an increased hemichannel function.

    Directory of Open Access Journals (Sweden)

    Yiguo Sun

    Full Text Available Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40 have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca2+]o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.

  18. Identification and characterization of tomato mutants affected in the Rx-mediated resistance to PVX isolates.

    Science.gov (United States)

    Sturbois, Bénédicte; Dubrana-Ourabah, Marie-Pierre; Gombert, Julie; Lasseur, Bertrand; Macquet, Audrey; Faure, Chantal; Bendahmane, Abdelhafid; Baurès, Isabelle; Candresse, Thierry

    2012-03-01

    Five tomato mutants affected in the Rx-mediated resistance against Potato virus X (PVX) were identified by screening a mutagenized population derived from a transgenic, Rx1-expressing 'Micro-Tom' line. Contrary to their parental line, they failed to develop lethal systemic necrosis upon infection with the virulent PVX-KH2 isolate. Sequence analysis and quantitative reverse-transcription polymerase chain reaction experiments indicated that the mutants are not affected in the Rx1 transgene or in the Hsp90, RanGap1 and RanGap2, Rar1 and Sgt1 genes. Inoculation with the PVX-CP4 avirulent isolate demonstrated that the Rx1 resistance was still effective in the mutants. In contrast, the virulent PVX-KH2 isolate accumulation was readily detectable in all mutants, which could further be separated in two groups depending on their ability to restrict the accumulation of PVX-RR, a mutant affected at two key positions for Rx1 elicitor activity. Finally, transient expression of the viral capsid protein elicitor indicated that the various mutants have retained the ability to mount an Rx1-mediated hypersensitive response. Taken together, the results obtained are consistent with a modification of the specificity or intensity of the Rx1-mediated response. The five Micro-Tom mutants should provide very valuable resources for the identification of novel tomato genes affecting the functioning of the Rx gene.

  19. Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

    Directory of Open Access Journals (Sweden)

    Sho W Suzuki

    2011-02-01

    Full Text Available Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA. We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

  20. Isolation, characterization, and mapping of the stay green mutant in rice.

    Science.gov (United States)

    Cha, K.-W.; Lee, Y.-J.; Koh, H.-J.; Lee, B.-M.; Nam, Y.-W.; Paek, N.-C.

    2002-03-01

    Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong- wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively.