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Sample records for mutagenesis reveal information

  1. Novel structure--function information on biogenic amine transporters revealed by site-directed mutagenesis and alkylation.

    PubMed

    Reith, Maarten E A

    2013-07-01

    The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET.

  2. Site directed mutagenesis of StSUT1 reveals target amino acids of regulation and stability.

    PubMed

    Krügel, Undine; Wiederhold, Elena; Pustogowa, Jelena; Hackel, Aleksandra; Grimm, Bernhard; Kühn, Christina

    2013-11-01

    Plant sucrose transporters (SUTs) are functional as sucrose-proton-cotransporters with an optimal transport activity in the acidic pH range. Recently, the pH optimum of the Solanum tuberosum sucrose transporter StSUT1 was experimentally determined to range at an unexpectedly low pH of 3 or even below. Various research groups have confirmed these surprising findings independently and in different organisms. Here we provide further experimental evidence for a pH optimum at physiological extrema. Site directed mutagenesis provides information about functional amino acids, which are highly conserved and responsible for this extraordinary increase in transport capacity under extreme pH conditions. Redox-dependent dimerization of the StSUT1 protein was described earlier. Here the ability of StSUT1 to form homodimers was demonstrated by heterologous expression in Lactococcus lactis and Xenopus leavis using Western blots, and in plants by bimolecular fluorescence complementation. Mutagenesis of highly conserved cysteine residues revealed their importance in protein stability. The accessibility of regulatory amino acid residues in the light of StSUT1's compartmentalization in membrane microdomains is discussed.

  3. Structure of the sodium channel pore revealed by serial cysteine mutagenesis.

    PubMed Central

    Pérez-García, M T; Chiamvimonvat, N; Marban, E; Tomaselli, G F

    1996-01-01

    The pores of voltage-gated cation channels are formed by four intramembrane segments that impart selectivity and conductance. Remarkably little is known about the higher order structure of these critical pore-lining or P segments. Serial cysteine mutagenesis reveals a pattern of side-chain accessibility that contradicts currently favored structural models based on alpha-helices or beta-strands. Like the active sites of many enzymes of known structure, the sodium channel pore consists of irregular loop regions. Images Fig. 1 Fig. 4 PMID:8552626

  4. Information Privacy Revealed

    ERIC Educational Resources Information Center

    Lavagnino, Merri Beth

    2013-01-01

    Why is Information Privacy the focus of the January-February 2013 issue of "EDUCAUSE Review" and "EDUCAUSE Review Online"? Results from the 2012 annual survey of the International Association of Privacy Professionals (IAPP) indicate that "meeting regulatory compliance requirements continues to be the top perceived driver…

  5. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

    PubMed Central

    Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  6. Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

    PubMed Central

    Ma, Li-Juan; Ohmert, Iris; Vardanyan, Vitya

    2011-01-01

    Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regularities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker, no correlation between V1/2 and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental findings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE β-subunits. PMID:21320432

  7. Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference.

    PubMed

    Mahan, Sheri D; Ireton, Greg C; Stoddard, Barry L; Black, Margaret E

    2004-07-20

    Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.

  8. Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

    PubMed Central

    Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

    2015-01-01

    A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

  9. In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication

    PubMed Central

    Wanrooij, Sjoerd; Miralles Fusté, Javier; Stewart, James B; Wanrooij, Paulina H; Samuelsson, Tore; Larsson, Nils-Göran; Gustafsson, Claes M; Falkenberg, Maria

    2012-01-01

    The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand-displacement model states that lagging-strand DNA synthesis is initiated from the origin of light-strand DNA replication (OriL), whereas the strand-coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand-displacement model for mtDNA replication. PMID:23090476

  10. Comprehensive cysteine-scanning mutagenesis reveals Claudin-2 pore-lining residues with different intrapore locations.

    PubMed

    Li, Jiahua; Zhuo, Min; Pei, Lei; Rajagopal, Madhumitha; Yu, Alan S L

    2014-03-07

    The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. We aimed to map the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutations within the ECL1 by expression in polyclonal Madin-Darby canine kidney II Tet-Off cells and found nine mutants that displayed a significant decrease of conductance after treatment with the thiol-reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate, indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal Madin-Darby canine kidney I Tet-Off cells and exposed them to thiol-reactive reagents. The maximum degree of inhibition of conductance, size selectivity of degree of inhibition, and size dependence of the kinetics of reaction were used to deduce the location of residues within the pore. Our data support the following sequence of pore-lining residues located from the narrowest to the widest part of the pore: Ser(68), Ser(47), Thr(62)/Ile(66), Thr(56), Thr(32)/Gly(45), and Met(52). The paracellular pore appears to primarily be lined by polar side chains, as expected for a predominantly aqueous environment. Furthermore, our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp(65)-Ser(68) that form a major part of the lining of the pore.

  11. Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis.

    PubMed

    Jin, Liang; Wu, Xuri; Xue, Yanjiu; Jin, Yue; Wang, Shuzhen; Chen, Yijun

    2017-02-15

    Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs.

  12. Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

    PubMed Central

    Soshilov, Anatoly A.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transformation using a panel of 12 AhR ligands. These ligands could be categorized into four distinct structurally related groups based on their ability to activate AhR mutants at position 319 in vitro. The mutation I319K was selectively activated by FICZ and not by other examined ligands in vitro and in cell culture. F318L and F318A mutations resulted in the conversion of AhR agonists β-naphthoflavone and 3-methylcholanthrene, respectively, into partial agonists/antagonists. Hsp90 binding to the AhR was decreased with several mutations and was inversely correlated with AhR ligand-binding promiscuity. Together, these data define overlapping amino acid residues within the AhR LBD involved in the selectivity of ligand binding, the agonist or antagonist mode of ligand binding, and hsp90 binding and provide insights into the ligand diversity of AhR activators. PMID:24591650

  13. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma.

    PubMed

    Mann, Karen M; Ward, Jerrold M; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W; Black, Michael A; Brett, Benjamin T; Sheetz, Todd E; Dupuy, Adam J; Chang, David K; Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Grimmond, Sean M; Rust, Alistair G; Adams, David J; Jenkins, Nancy A; Copeland, Neal G

    2012-04-17

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.

  14. Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels.

    PubMed

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K; Ramírez, David; Netter, Michael F; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-22

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels.

  15. Alanine scanning mutagenesis of anti-TRAP (AT) reveals residues involved in binding to TRAP.

    PubMed

    Chen, Yanling; Gollnick, Paul

    2008-04-11

    The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic (trp) genes in response to changes in intracellular levels of free l-tryptophan in many Gram-positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT(3)) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT(12)) structure. Using alanine-scanning mutagenesis we found four residues, all located on the "top" region of AT(3), that are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation.

  16. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation.

    PubMed

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-05-07

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

  17. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

    PubMed Central

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-01-01

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy. PMID:25950479

  18. Reaction Mechanism of Glutamate Carboxypeptidase II Revealed by Mutagenesis, X-ray Crystallography, and Computational Methods

    SciTech Connect

    Klusak, Vojtech; Barinka, Cyril; Plechanovova, Anna; Mlcochova, Petra; Konvalinka, Jan; Rulisek, Lubomir; Lubkowski, Jacek

    2009-05-29

    Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 {angstrom} resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be {Delta}G {approx} 22({+-}5) kcal{center_dot}mol{sup -1}, which is in a good agreement with the experimentally observed reaction rate constant (k{sub cat} {approx} 1 s{sup -1}). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.

  19. Role of the "helix clamp" in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis.

    PubMed

    Beard, W A; Minnick, D T; Wade, C L; Prasad, R; Won, R L; Kumar, A; Kunkel, T A; Wilson, S H

    1996-05-24

    Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the "helix clamp" (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template.primer (T.P) binding as revealed by a lack of an effect on Km, T.P, Ki for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, koff, T.P and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T.P (L282A and R284A) demonstrated that these mutant enzymes could bind T.P stoichiometrically and tightly. In contrast, active site concentrations determined from "burst" experiments suggest that the lower activity is due to a smaller populations of enzyme bound productively to T.P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3

  20. Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function

    PubMed Central

    Liu, Yang; Kretz, Colin A.; Maeder, Morgan L.; Richter, Catherine E.; Tsao, Philip; Vo, Andy H.; Huarng, Michael C.; Rode, Thomas; Hu, Zhilian; Mehra, Rohit; Olson, Steven T.; Joung, J. Keith

    2014-01-01

    Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis. PMID:24782510

  1. Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance

    PubMed Central

    Yung, Mimi C.; Park, Dan M.; Overton, K. Wesley; Blow, Matthew J.; Hoover, Cindi A.; Smit, John; Murray, Sean R.; Ricci, Dante P.; Christen, Beat; Bowman, Grant R.

    2015-01-01

    ABSTRACT The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus

  2. Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, QM/MM Simulation, and Mutagenesis

    PubMed Central

    2014-01-01

    N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C

  3. Genome-Wide Mouse Mutagenesis Reveals CD45-Mediated T Cell Function as Critical in Protective Immunity to HSV-1

    PubMed Central

    Caignard, Grégory; Leiva-Torres, Gabriel A.; Leney-Greene, Michael; Charbonneau, Benoit; Dumaine, Anne; Fodil-Cornu, Nassima; Pyzik, Michal; Cingolani, Pablo; Schwartzentruber, Jeremy; Dupaul-Chicoine, Jeremy; Guo, Huaijian; Saleh, Maya; Veillette, André; Lathrop, Marc; Blanchette, Mathieu; Majewski, Jacek; Pearson, Angela; Vidal, Silvia M.

    2013-01-01

    Herpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (PtprcL3X), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected PtprcL3X mice accounting for hyper-inflammation and pathological damages caused by viral replication. PtprcL3X mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into PtprcL3X mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4+ and CD8+ T cells and could be attributed to function of CD4+ T helper 1 (Th1) cells in CD8+ T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent

  4. The role of 23S ribosomal RNA residue A2451 in peptide bond synthesis revealed by atomic mutagenesis.

    PubMed

    Lang, Kathrin; Erlacher, Matthias; Wilson, Daniel N; Micura, Ronald; Polacek, Norbert

    2008-05-01

    Peptide bond formation is a fundamental reaction in biology, catalyzed by the ribosomal peptidyl-transferase ribozyme. Although all active-site 23S ribosomal RNA nucleotides are universally conserved, atomic mutagenesis suggests that these nucleobases do not carry functional groups directly involved in peptide bond formation. Instead, a single ribose 2'-hydroxyl group at A2451 was identified to be of pivotal importance. Here, we altered the chemical characteristics by replacing its 2'-hydroxyl with selected functional groups and demonstrate that hydrogen donor capability is essential for transpeptidation. We propose that the A2451-2'-hydroxyl directly hydrogen bonds to the P-site tRNA-A76 ribose. This promotes an effective A76 ribose C2'-endo conformation to support amide synthesis via a proton shuttle mechanism. Simultaneously, the direct interaction of A2451 with A76 renders the intramolecular transesterification of the peptide from the 3'- to 2'-oxygen unfeasible, thus promoting effective peptide bond synthesis.

  5. Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia

    PubMed Central

    Tang, Jian Zhong; Carmichael, Catherine L.; Shi, Wei; Metcalf, Donald; Ng, Ashley P.; Hyland, Craig D.; Jenkins, Nancy A.; Copeland, Neal G.; Howell, Viive M.; Zhao, Zhizhuang Joe; Smyth, Gordon K.; Kile, Benjamin T.; Alexander, Warren S.

    2013-01-01

    To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG–induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG–induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG–driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals. PMID:23533276

  6. Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia.

    PubMed

    Tang, Jian Zhong; Carmichael, Catherine L; Shi, Wei; Metcalf, Donald; Ng, Ashley P; Hyland, Craig D; Jenkins, Nancy A; Copeland, Neal G; Howell, Viive M; Zhao, Zhizhuang Joe; Smyth, Gordon K; Kile, Benjamin T; Alexander, Warren S

    2013-04-09

    To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG-induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG-induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG-driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals.

  7. CRISPR/Cas9 Mutagenesis Reveals Versatile Roles of Hox Genes in Crustacean Limb Specification and Evolution.

    PubMed

    Martin, Arnaud; Serano, Julia M; Jarvis, Erin; Bruce, Heather S; Wang, Jennifer; Ray, Shagnik; Barker, Carryn A; O'Connell, Liam C; Patel, Nipam H

    2016-01-11

    Crustaceans possess a diverse array of specialized limbs. Although shifts in Hox gene expression domains have been postulated to play a role in generating this limb diversity, little functional data have been provided to understand the precise roles of Hox genes during crustacean development. We used a combination of CRISPR/Cas9-targeted mutagenesis and RNAi knockdown to decipher the function of the six Hox genes expressed in the developing mouth and trunk of the amphipod Parhyale hawaiensis. These experimentally manipulated animals display specific and striking homeotic transformations. We found that abdominal-A (abd-A) and Abdominal-B (Abd-B) are required for proper posterior patterning, with knockout of Abd-B resulting in an animal with thoracic type legs along what would have been an abdomen, and abd-A disruption generating a simplified body plan characterized by a loss of specialization in both abdominal and thoracic appendages. In the thorax, Ubx is necessary for gill development and for repression of gnathal fate, and Antp dictates claw morphology. In the mouth, Scr and Antp confer the part-gnathal, part-thoracic hybrid identity of the maxilliped, and Scr and Dfd prevent antennal identity in posterior head segments. Our results allow us to define the role Hox genes play in specifying each appendage type in Parhyale, including the modular nature by which some appendages are patterned by Hox gene inputs. In addition, we define how changes in Hox gene expression have generated morphological differences between crustacean species. Finally, we also highlight the utility of CRISPR/Cas9-based somatic mutagenesis in emerging model organisms.

  8. Hiding personal information reveals the worst

    PubMed Central

    John, Leslie K.; Barasz, Kate; Norton, Michael I.

    2016-01-01

    Seven experiments explore people’s decisions to share or withhold personal information, and the wisdom of such decisions. When people choose not to reveal information—to be “hiders”—they are judged negatively by others (experiment 1). These negative judgments emerge when hiding is volitional (experiments 2A and 2B) and are driven by decreases in trustworthiness engendered by decisions to hide (experiments 3A and 3B). Moreover, hiders do not intuit these negative consequences: given the choice to withhold or reveal unsavory information, people often choose to withhold, but observers rate those who reveal even questionable behavior more positively (experiments 4A and 4B). The negative impact of hiding holds whether opting not to disclose unflattering (drug use, poor grades, and sexually transmitted diseases) or flattering (blood donations) information, and across decisions ranging from whom to date to whom to hire. When faced with decisions about disclosure, decision-makers should be aware not just of the risk of revealing, but of what hiding reveals. PMID:26755591

  9. Site-directed mutagenesis of a tetrameric dandelion polyphenol oxidase (PPO-6) reveals the site of subunit interaction.

    PubMed

    Dirks-Hofmeister, Mareike E; Inlow, Jennifer K; Moerschbacher, Bruno M

    2012-09-01

    Polyphenol oxidases (PPOs) catalyze the oxidation of ortho-diphenols to the corresponding quinones (EC 1.10.3.1). In plants PPOs appear in gene families, and the corresponding isoenzymes are located to the thylakoid lumen of chloroplasts. Although plant PPOs are often discussed with regard to their role in defense reactions, a common physiological function has not yet been defined. We analyzed a tetrameric PPO isoenzyme (PPO-6) from dandelion (Taraxacum officinale) heterologously expressed in Escherichia coli, and found it to display cooperativity in catalysis, a phenomenon that has rarely been shown for plant PPOs previously. The identification of a surface-exposed cysteine (197) through molecular modeling followed by site-directed mutagenesis proved this amino acid residue to stabilize the tetramer via a disulfide linkage. The C197S-mutein still forms a tetrameric structure but shows impaired enzymatic efficiency and cooperativity and a reduction in stability. These findings indicate that oligomerization may be a physiological requirement for PPO-6 stability and function in vivo and raise new questions regarding distinct functions for specific PPO isoenzymes in plants.

  10. Saturation Mutagenesis of the HIV-1 Envelope CD4 Binding Loop Reveals Residues Controlling Distinct Trimer Conformations

    PubMed Central

    Bolon, Daniel; Clapham, Paul R.

    2016-01-01

    The conformation of HIV-1 envelope (Env) glycoprotein trimers is key in ensuring protection against waves of neutralizing antibodies generated during infection, while maintaining sufficient exposure of the CD4 binding site (CD4bs) for viral entry. The CD4 binding loop on Env is an early contact site for CD4 while penetration of a proximal cavity by CD4 triggers Env conformational changes for entry. The role of residues in the CD4 binding loop in regulating the conformation of the trimer and trimer association domain (TAD) was investigated using a novel saturation mutagenesis approach. Single mutations identified, resulted in distinct trimer conformations affecting CD4bs exposure, the glycan shield and the TAD across diverse HIV-1 clades. Importantly, mutations that improve access to the CD4bs without exposing the immunodominant V3 loop were identified. The different trimer conformations identified will affect the specificity and breadth of nabs elicited in vivo and are important to consider in design of Env immunogens for vaccines. PMID:27820858

  11. Functional mutagenesis screens reveal the ‘cap structure’ formation in disulfide-bridge free TASK channels

    PubMed Central

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K.; Ramírez, David; Netter, Michael F.; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-01

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels. PMID:26794006

  12. Site-specific Mtm1 mutagenesis by an AAV-Cre vector reveals that myotubularin is essential in adult muscle.

    PubMed

    Joubert, Romain; Vignaud, Alban; Le, Mickaël; Moal, Christelle; Messaddeq, Nadia; Buj-Bello, Anna

    2013-05-01

    Manipulation of the mouse genome by site-specific mutagenesis has been extensively used to study gene function and model human disorders. Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle. However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue in the context of not only muscle biology but also therapies. To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter. We report that a single injection of this vector into muscles of 3-month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning, such as mitochondria and nuclei, T-tubule defects and severe muscle weakness. In addition, our results show that MTM1-related atrophy and dysfunction correlate with abnormalities in satellite cell number and markers of autophagy, protein synthesis and neuromuscular junction transmission. The expression level of atrogenes was also analyzed. Therefore, we provide a valuable tissue model that recapitulates the main features of the disease, and it is useful to study pathogenesis and evaluate therapeutic strategies. We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, suggesting that therapies will be required for the entire life of XLMTM patients.

  13. Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

    PubMed Central

    2010-01-01

    Background Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility. Results We molecularly analyzed a novel set of induced barley mlo mutants and used site-directed mutagenesis in combination with transient gene expression to unravel novel amino acid residues of functional significance. We integrate these results with previous findings to map functionally important regions of the heptahelical Mlo protein. Our data reveal the second and third cytoplasmic loop as being particularly sensitive to functional impediment by mutational perturbation, suggesting that these regions are critical for the susceptibility-conferring activity of the Mlo protein. In contrast, only mutations in the second but not the third cytoplasmic loop appear to trigger the Endoplasmic Reticulum-localized quality control machinery that ensures the biogenesis of properly folded membrane proteins. Conclusion Our findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control. PMID:20170486

  14. Mutagenesis of GATA motifs controlling the endoderm regulator elt-2 reveals distinct dominant and secondary cis-regulatory elements.

    PubMed

    Du, Lawrence; Tracy, Sharon; Rifkin, Scott A

    2016-04-01

    Cis-regulatory elements (CREs) are crucial links in developmental gene regulatory networks, but in many cases, it can be difficult to discern whether similar CREs are functionally equivalent. We found that despite similar conservation and binding capability to upstream activators, different GATA cis-regulatory motifs within the promoter of the C. elegans endoderm regulator elt-2 play distinctive roles in activating and modulating gene expression throughout development. We fused wild-type and mutant versions of the elt-2 promoter to a gfp reporter and inserted these constructs as single copies into the C. elegans genome. We then counted early embryonic gfp transcripts using single-molecule RNA FISH (smFISH) and quantified gut GFP fluorescence. We determined that a single primary dominant GATA motif located 527bp upstream of the elt-2 start codon was necessary for both embryonic activation and later maintenance of transcription, while nearby secondary GATA motifs played largely subtle roles in modulating postembryonic levels of elt-2. Mutation of the primary activating site increased low-level spatiotemporally ectopic stochastic transcription, indicating that this site acts repressively in non-endoderm cells. Our results reveal that CREs with similar GATA factor binding affinities in close proximity can play very divergent context-dependent roles in regulating the expression of a developmentally critical gene in vivo.

  15. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  16. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGES

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  17. Site-directed mutagenesis reveals regions implicated in the stability and fiber formation of human λ3r light chains.

    PubMed

    Villalba, Miryam I; Canul-Tec, Juan C; Luna-Martínez, Oscar D; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A; Becerril, Baltazar

    2015-01-30

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40-60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL.

  18. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

    SciTech Connect

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2014-12-11

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this paper, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. Finally, this mutagenic approach helped to identify key regions implicated in λ3 AL.

  19. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

    DOE PAGES

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; ...

    2014-12-11

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this paper, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, themore » second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. Finally, this mutagenic approach helped to identify key regions implicated in λ3 AL.« less

  20. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    PubMed Central

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  1. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  2. Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation.

    PubMed

    Li, Jinyun; Wang, Nian

    2011-01-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and

  3. Genome-Wide Mutagenesis of Xanthomonas axonopodis pv. citri Reveals Novel Genetic Determinants and Regulation Mechanisms of Biofilm Formation

    PubMed Central

    Li, Jinyun; Wang, Nian

    2011-01-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and

  4. Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site-directed mutagenesis

    PubMed Central

    Roulias, A; Pichler, U; Hauser, M; Himly, M; Hofer, H; Lackner, P; Ebner, C; Briza, P; Bohle, B; Egger, M; Wallner, M; Ferreira, F

    2014-01-01

    Background Birch pollen allergies are frequently associated with adverse reactions to various fruits, nuts, or vegetables, described as pollen–food syndrome (PFS) and caused by cross-reactive IgE antibodies primarily directed against Bet v 1. Specific immunotherapy (SIT) represents an effective treatment for inhalant allergies; however, successful birch pollen SIT does not correlate well with the amelioration of concomitant food allergies. Methods As vaccine candidates, apple Mal d 1 as well as hazelnut Cor a 1 derivatives were designed by in silico backbone analyses of the respective allergens. The proteins were produced by site-directed mutagenesis as fold variants of their parental allergens. Because Mal d 1 and Cor a 1 form cysteine-mediated aggregates, nonaggregative cysteine to serine mutants were also generated. The proteins were characterized physicochemically, immunologically, and in in vivo models with or without adjuvant. Results The structurally modified proteins showed significantly decreased IgE binding capacity. Notably, both in vivo models revealed reduced immunogenicity of the hypoallergenic fold variants. When formulated with alum, the monomeric cysteine mutants induced a similar immune response as the aggregated parental allergens, which is in contrast with data published on Bet v 1. Conclusion These findings lead to the suggestion that the Bet v 1 structure has unique intrinsic properties, which could account for its high allergenicity. Obviously, these characteristics are not entirely shared with its food homologues from apple and hazelnut. Thus, it is important to tackle pollen-related food allergies from different angles for the generation of effective vaccine candidates to treat birch PFS. PMID:24224690

  5. Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

    PubMed

    Yoshida, Keita; Hozumi, Akiko; Treen, Nicholas; Sakuma, Tetsushi; Yamamoto, Takashi; Shirae-Kurabayashi, Maki; Sasakura, Yasunori

    2017-03-15

    The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.

  6. Significant differences in cell-cell fusion and viral entry between strains revealed by scanning mutagenesis of the C-heptad repeat of HIV gp41.

    PubMed

    Diaz-Aguilar, Barbara; Dewispelaere, Karen; Yi, Hyun Ah; Jacobs, Amy

    2013-05-21

    The transmembrane subunit, gp41, of the HIV envelope mediates the viral fusion step of entry into the host cell. The protein consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain contains a fusion peptide, an N-terminal heptad repeat, a loop region, a C-terminal heptad repeat (CHR), and a membrane-proximal external region. For this study, we examined each amino acid in the CHR (residues 623-659) by alanine scanning mutagenesis in two HIV strains: one CCR5-utilizing strain (JRFL) and one CXCR4-utilizing strain (HXB2). We studied the functional importance of each amino acid residue by measuring mutational effects in both cell-cell fusion and viral entry and assessing envelope expression and gp120-gp41 proteolytic processing. The transmembrane subunit of the HIV envelope, gp41, is very sensitive to subtle changes, like alanine substitution, which severely affect envelope function at multiple sites. Two important general findings are apparent when the entire data set from this study is taken into account. (1) Strain HXB2 is much more stable to mutagenesis than strain JRFL, and (2) viral entry is much more stable to mutagenesis than cell-cell fusion. These findings strengthen our notion that gp41 is a vulnerable target for therapeutic and prophylactic intervention. Further structural studies aimed at gaining a full understanding of the intermediate states that drive HIV membrane fusion are imperative.

  7. Readaptation of a low-virulence influenza H9 escape mutant in mice: the role of changes in hemagglutinin as revealed by site-specific mutagenesis.

    PubMed

    Ilyushina, N A; Rudneva, I A; Khalenkov, A M; Timofeeva, T A; Krylov, P S; Webster, R G; Kaverin, N V

    2010-01-01

    In our earlier studies, we showed that an escape mutant of mouse-adapted H9N2 influenza virus carrying a T198N amino acid change in heamagglutinin (HA) has a lowered virulence for mice. The readaptation of this mutant to mice was associated with N198S or N198D reverse mutations. In this study, single-gene reassortants having HA gene of the wild-type virus, its low-virulence escape mutant, or a readapted variant were generated by site-specific mutagenesis and assayed for virulence. The results showed that antibody-selected mutations in the HA of H9 influenza virus can decrease mortality and virus accumulation in mouse lungs, though not in nasal turbinates, and the effect may be compensated by reverse mutations in the course of passaging.

  8. Mutagenesis of lysines 156 and 159 in human immunodeficiency virus type 1 integrase (IN) reveals differential interactions between these residues and different IN inhibitors.

    PubMed

    Crosby, David C; Lei, Xiangyang; Gibbs, Charles G; Reinecke, Manfred G; Robinson, W Edward

    2015-01-01

    Human immunodeficiency virus (HIV) type I integrase (IN) active site, and viral DNA-binding residues K156 and K159 are predicted to interact both with strand transfer-selective IN inhibitors (STI), e.g. L-731,988, Elvitegravir (EVG), and the FDA-approved IN inhibitor, Raltegravir (RGV), and strand transfer non-selective inhibitors, e.g. dicaffeoyltartaric acids (DCTAs), e.g. L-chicoric acid (L-CA). To test posited roles for these two lysine residues in inhibitor action we assayed the potency of L-CA and several STI against a panel of K156 and K159 mutants. Mutagenesis of K156 conferred resistance to L-CA and mutagenesis of either K156 or K159 conferred resistance to STI indicating that the cationic charge at these two viral DNA-binding residues is important for inhibitor potency. IN K156N, a reported polymorphism associated with resistance to RGV, conferred resistance to L-CA and STI as well. To investigate the apparent preference L-CA exhibits for interactions with K156, we assayed the potency of several hybrid inhibitors containing combinations of DCTA and STI pharmacophores against recombinant IN K156A or K159A. Although K156A conferred resistance to diketo acid-branched bis-catechol hybrid inhibitors, neither K156A nor K159A conferred resistance to their monocatechol counterparts, suggesting that bis-catechol moieties direct DCTAs toward K156. In contrast, STI were more promiscuous in their interaction with K156 and K159. Taken together, the results of this study indicate that DCTAs interact with IN in a manner different than that of STI and suggest that DCTAs are an attractive candidate chemotype for development into drugs potent against STI-resistant IN.

  9. A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

    PubMed Central

    Redding, K; MacMillan, F; Leibl, W; Brettel, K; Hanley, J; Rutherford, A W; Breton, J; Rochaix, J D

    1998-01-01

    The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls. PMID:9427740

  10. Large-scale transposon mutagenesis of Photobacterium profundum SS9 reveals new genetic loci important for growth at low temperature and high pressure.

    PubMed

    Lauro, Federico M; Tran, Khiem; Vezzi, Alessandro; Vitulo, Nicola; Valle, Giorgio; Bartlett, Douglas H

    2008-03-01

    Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacterium Photobacterium profundum strain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.

  11. Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy.

    PubMed

    Perez, H D; Vilander, L; Andrews, W H; Holmes, R

    1994-09-09

    Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.

  12. A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene

    PubMed Central

    Yin, Bin; Delwel, Ruud; Valk, Peter J.; Wallace, Margaret R.; Loh, Mignon L.; Shannon, Kevin M.

    2009-01-01

    NF1 inactivation occurs in specific human cancers, including juvenile myelomonocytic leukemia, an aggressive myeloproliferative disorder of childhood. However, evidence suggests that Nf1 loss alone does not cause leukemia. We therefore hypothesized that inactivation of the Nf1 tumor suppressor gene requires cooperating mutations to cause acute leukemia. To search for candidate genes that cooperate with Nf1 deficiency in leukemogenesis, we performed a forward genetic screen using retroviral insertion mutagenesis in Nf1 mutant mice. We identified 43 common proviral insertion sites that contain candidate genes involved in leukemogenesis. One of these genes, Bcl11a, confers a growth advantage in cultured Nf1 mutant hematopoietic cells and causes early onset of leukemia of either myeloid or lymphoid lineage in mice when expressed in Nf1-deficient bone marrow. Bcl11a-expressing cells display compromised p21Cip1 induction, suggesting that Bcl11a's oncogenic effects are mediated, in part, through suppression of p21Cip1. Importantly, Bcl11a is expressed in human chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia samples. A subset of AML patients, who had poor outcomes, of 16 clusters, displayed high levels of BCL11A in leukemic cells. These findings suggest that deregulated Bcl11a cooperates with Nf1 in leukemogenesis, and a therapeutic strategy targeting the BCL11A pathway may prove beneficial in the treatment of leukemia. PMID:18948576

  13. Structure-Function Analysis of Porcine Cytochrome P450 3A29 in the Hydroxylation of T-2 Toxin as Revealed by Docking and Mutagenesis Studies

    PubMed Central

    Wang, Xu; Ma, Hongmin; Pan, Yuanhu; Huang, Lingli; Hao, Haihong; Dai, Menghong; Yuan, Zonghui

    2014-01-01

    T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29) played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CLint value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CLint values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3′-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3′-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin. PMID:25184434

  14. Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding.

    PubMed

    Gkountelias, Kostas; Tselios, Theodoros; Venihaki, Maria; Deraos, George; Lazaridis, Iakovos; Rassouli, Olga; Gravanis, Achille; Liapakis, George

    2009-04-01

    Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF(1)), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF(1) we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF(1). The W259A, F260A, and W259A/F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF(1) compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF(1). This is the first time that a specific region of CRF(1) has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.

  15. Computer Simulation of Mutagenesis.

    ERIC Educational Resources Information Center

    North, J. C.; Dent, M. T.

    1978-01-01

    A FORTRAN program is described which simulates point-substitution mutations in the DNA strands of typical organisms. Its objective is to help students to understand the significance and structure of the genetic code, and the mechanisms and effect of mutagenesis. (Author/BB)

  16. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  17. Mechanism of proflavin mutagenesis.

    PubMed

    Sarabhai, A; Lamfrom, H

    1969-08-01

    The mutagenic action of proflavin on bacteriophage T4 is greater in the presence of defective T4 ligase than in the presence of normal T4 ligase. This suggests that the persistence of single-strand breaks in DNA enhances proflavin mutagenesis.

  18. ID-Check: Online Concealed Information Test Reveals True Identity.

    PubMed

    Verschuere, Bruno; Kleinberg, Bennett

    2016-01-01

    The Internet has already changed people's lives considerably and is likely to drastically change forensic research. We developed a web-based test to reveal concealed autobiographical information. Initial studies identified a number of conditions that affect diagnostic efficiency. By combining these moderators, this study investigated the full potential of the online ID-check. Participants (n = 101) tried to hide their identity and claimed a false identity in a reaction time-based Concealed Information Test. Half of the participants were presented with personal details (e.g., first name, last name, birthday), whereas the others only saw irrelevant details. Results showed that participants' true identity could be detected with high accuracy (AUC = 0.98; overall accuracy: 86-94%). Online memory detection can reliably and validly detect whether someone is hiding their true identity. This suggests that online memory detection might become a valuable tool for forensic applications.

  19. Lethal Mutagenesis Failure May Augment Viral Adaptation

    PubMed Central

    Paff, Matthew L.; Stolte, Steven P.; Bull, James J.

    2014-01-01

    Lethal mutagenesis, the attempt to extinguish a population by elevating its mutation rate, has been endorsed in the virology literature as a promising approach for treating viral infections. In support of the concept, in vitro studies have forced viral extinction with high doses of mutagenic drugs. However, the one known mutagenic drug used on patients commonly fails to cure infections, and in vitro studies typically find a wide range of mutagenic conditions permissive for viral growth. A key question becomes how subsequent evolution is affected if the viral population is mutated but avoids extinction—Is viral adaptation augmented rather than suppressed? Here we consider the evolution of highly mutated populations surviving mutagenesis, using the DNA phage T7. In assays using inhibitory hosts, whenever resistance mutants were observed, the mutagenized populations exhibited higher frequencies, but some inhibitors blocked plaque formation by even the mutagenized stock. Second, outgrowth of previously mutagenized populations led to rapid and potentially complete fitness recovery but polymorphism was slow to decay, and mutations exhibited inconsistent patterns of change. Third, the combination of population bottlenecks with mutagenesis did cause fitness declines, revealing a vulnerability that was not apparent from mutagenesis of large populations. The results show that a population surviving high mutagenesis may exhibit enhanced adaptation in some environments and experience little negative fitness consequences in many others. PMID:24092771

  20. Systematic mutagenesis of all predicted gntR genes in Xanthomonas campestris pv. campestris reveals a GntR family transcriptional regulator controlling hypersensitive response and virulence.

    PubMed

    An, Shi-Qi; Lu, Guang-Tao; Su, Hui-Zhao; Li, Rui-Fang; He, Yong-Qiang; Jiang, Bo-Le; Tang, Dong-Jie; Tang, Ji-Liang

    2011-09-01

    The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.

  1. Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase

    PubMed Central

    Dellas, Nikki; Snyder, Jamie C.; Dills, Michael; Nicolay, Sheena J.; Kerchner, Keshia M.; Brumfield, Susan K.; Lawrence, C. Martin

    2015-01-01

    ABSTRACT Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. IMPORTANCE STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives

  2. Mutagenesis of hetR reveals amino acids necessary for HetR function in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Risser, Douglas D; Callahan, Sean M

    2007-03-01

    HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking hetR on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het- phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type hetR under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a hetR deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het- phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in Anabaena sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.

  3. Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

    SciTech Connect

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A.

    2014-10-02

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  4. A comprehensive alanine-scanning mutagenesis study reveals roles for salt bridges in the structure and activity of Pseudomonas aeruginosa elastase.

    PubMed

    Bian, Fei; Yue, Shousong; Peng, Zhenying; Zhang, Xiaowei; Chen, Gao; Yu, Jinhui; Xuan, Ning; Bi, Yuping

    2015-01-01

    The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (kcat/Km), half-lives (t1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.

  5. Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion.

    PubMed

    Bose, Sayantan; Welch, Brett D; Kors, Christopher A; Yuan, Ping; Jardetzky, Theodore S; Lamb, Robert A

    2011-12-01

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  6. Individual olfactory perception reveals meaningful nonolfactory genetic information.

    PubMed

    Secundo, Lavi; Snitz, Kobi; Weissler, Kineret; Pinchover, Liron; Shoenfeld, Yehuda; Loewenthal, Ron; Agmon-Levin, Nancy; Frumin, Idan; Bar-Zvi, Dana; Shushan, Sagit; Sobel, Noam

    2015-07-14

    Each person expresses a potentially unique subset of ∼ 400 different olfactory receptor subtypes. Given that the receptors we express partially determine the odors we smell, it follows that each person may have a unique nose; to capture this, we devised a sensitive test of olfactory perception we termed the "olfactory fingerprint." Olfactory fingerprints relied on matrices of perceived odorant similarity derived from descriptors applied to the odorants. We initially fingerprinted 89 individuals using 28 odors and 54 descriptors. We found that each person had a unique olfactory fingerprint (P < 10(-10)), which was odor specific but descriptor independent. We could identify individuals from this pool using randomly selected sets of 7 odors and 11 descriptors alone. Extrapolating from this data, we determined that using 34 odors and 35 descriptors we could individually identify each of the 7 billion people on earth. Olfactory perception, however, fluctuates over time, calling into question our proposed perceptual readout of presumably stable genetic makeup. To test whether fingerprints remain informative despite this temporal fluctuation, building on the linkage between olfactory receptors and HLA, we hypothesized that olfactory perception may relate to HLA. We obtained olfactory fingerprints and HLA typing for 130 individuals, and found that olfactory fingerprint matching using only four odorants was significantly related to HLA matching (P < 10(-4)), such that olfactory fingerprints can save 32% of HLA tests in a population screen (P < 10(-6)). In conclusion, a precise measure of olfactory perception reveals meaningful nonolfactory genetic information.

  7. Revealing biological information using data structuring and automated learning.

    PubMed

    Mohorianu, Irina; Moulton, Vincent

    2010-11-01

    The intermediary steps between a biological hypothesis, concretized in the input data, and meaningful results, validated using biological experiments, commonly employ bioinformatics tools. Starting with storage of the data and ending with a statistical analysis of the significance of the results, every step in a bioinformatics analysis has been intensively studied and the resulting methods and models patented. This review summarizes the bioinformatics patents that have been developed mainly for the study of genes, and points out the universal applicability of bioinformatics methods to other related studies such as RNA interference. More specifically, we overview the steps undertaken in the majority of bioinformatics analyses, highlighting, for each, various approaches that have been developed to reveal details from different perspectives. First we consider data warehousing, the first task that has to be performed efficiently, optimizing the structure of the database, in order to facilitate both the subsequent steps and the retrieval of information. Next, we review data mining, which occupies the central part of most bioinformatics analyses, presenting patents concerning differential expression, unsupervised and supervised learning. Last, we discuss how networks of interactions of genes or other players in the cell may be created, which help draw biological conclusions and have been described in several patents.

  8. Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions

    PubMed Central

    Dietel, Kristin; Beator, Barbara; Dolgova, Olga; Fan, Ben; Bleiss, Wilfrid; Ziegler, Jörg; Schmid, Michael; Hartmann, Anton; Borriss, Rainer

    2014-01-01

    Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. PMID:24847778

  9. Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

    PubMed

    Sun, Zhoutong; Lonsdale, Richard; Li, Guangyue; Reetz, Manfred T

    2016-10-04

    Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.

  10. Transposon Mutagenesis in Mice

    PubMed Central

    Largaespada, David A.

    2010-01-01

    Understanding the functional landscape of the mammalian genome is the next big challenge of biomedical research. The completion of the first phases of the mouse and human genome projects, and expression analyses using microarray hybridization, generate critically important questions about the functional landscape and structure of the mammalian genome: how many genes, and of what type, are there; what kind of functional elements make up a properly functioning gene? One step in this process will be to create mutations in every identifiable mouse gene and analyze the resultant phenotypes. Transposons are being considered as tools to further initiatives to create a comprehensive resource of mutant mouse strains. Also, it may be possible to use transposons in true forward genetic screens in the mouse. The “Sleeping Beauty” (SB) transposon system is one such tool. Moreover, due to its tendency for local hopping, SB has been proposed as a method for regional saturation mutagenesis of the mouse genome. In this chapter, we review the tools and methods currently available to create mutant mice using in vivo, germline transposition in mice. PMID:19266336

  11. Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions Between NS2A and NS2B Transmembrane Segments.

    PubMed

    Wu, Ren-Huang; Tsai, Ming-Han; Tsai, Kuen-Nan; Tian, Jia Ni; Wu, Jian-Sung; Wu, Su-Ying; Chern, Jyh-Haur; Chen, Chun-Hong; Yueh, Andrew

    2017-04-05

    The NS2A protein of Dengue virus (DENV) has eight predicted transmembrane segments (pTMS1-8) and participates in RNA replication, virion assembly, and host antiviral response. However, the roles of specific amino acid residues within the pTMS regions of NS2A during the viral life cycle are not clear. Here, we explored the function of DENV NS2A by introducing a series of alanine substitutions into the N-terminal half (pTMS1-4) of the protein in the context of a DENV infectious clone or subgenomic replicon. Six NS2A mutants (NM5, 7, 9, and 17-19) around pTMS1-2 displayed a novel phenotype showing a >1000-fold reduction in virus yield, an absence of plaque formation despite wild-type-like replicon activity, and infectious virus-like particle yields. The HEK293 cells infected with those six NS2A mutant viruses failed to cause a virus-induced cytopathic effect (CPE) by MitoCapture staining, cell proliferation, and lactate dehydrogenase release assays. Sequencing analyses of pseudorevertant viruses derived from lethal mutant viruses revealed two consensus reversion mutations, leucine-to-phenylalanine at codon 181 (L181F) within the pTMS7 of NS2A and isoleucine-to-threonine at codon 114 (I114T) within NS2B. The introduction of NS2A-L181F mutation into the lethal (NM15, 16, 25, and 33) and CPE-defective (NM7, 9, and 19) mutants substantially rescued virus infectivity and virus-induced CPE, respectively, whereas NS2B-L114T mutation rescued NM16, 25, and 33 mutants. In conclusion, the results revealed the essential roles of the N-terminal half of NS2A in RNA replication and virus-induced CPE. Intramolecular interactions between pTMSs of NS2A and intermolecular interactions between NS2A and NS2B protein were also implicated.Importance: The characterization of the N-terminal (current study) and C-terminal half of DENV NS2A is the most comprehensive mutagenesis study to date to investigate the function of NS2A during the flaviviral life cycle. A novel region responsible for

  12. Revealing Relationships among Relevant Climate Variables with Information Theory

    NASA Technical Reports Server (NTRS)

    Knuth, Kevin H.; Golera, Anthony; Curry, Charles T.; Huyser, Karen A.; Kevin R. Wheeler; Rossow, William B.

    2005-01-01

    The primary objective of the NASA Earth-Sun Exploration Technology Office is to understand the observed Earth climate variability, thus enabling the determination and prediction of the climate's response to both natural and human-induced forcing. We are currently developing a suite of computational tools that will allow researchers to calculate, from data, a variety of information-theoretic quantities such as mutual information, which can be used to identify relationships among climate variables, and transfer entropy, which indicates the possibility of causal interactions. Our tools estimate these quantities along with their associated error bars, the latter of which is critical for describing the degree of uncertainty in the estimates. This work is based upon optimal binning techniques that we have developed for piecewise-constant, histogram-style models of the underlying density functions. Two useful side benefits have already been discovered. The first allows a researcher to determine whether there exist sufficient data to estimate the underlying probability density. The second permits one to determine an acceptable degree of round-off when compressing data for efficient transfer and storage. We also demonstrate how mutual information and transfer entropy can be applied so as to allow researchers not only to identify relations among climate variables, but also to characterize and quantify their possible causal interactions.

  13. Environmental stress induces trinucleotide repeat mutagenesis in human cells

    PubMed Central

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

    2015-01-01

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

  14. 41 CFR 102-81.30 - What information must job applicants at child care centers reveal?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... job applicants at child care centers reveal? 102-81.30 Section 102-81.30 Public Contracts and Property... PROPERTY 81-SECURITY Security § 102-81.30 What information must job applicants at child care centers reveal... on the job application. Employment at a child care facility means any position that involves...

  15. 41 CFR 102-81.30 - What information must job applicants at child care centers reveal?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... job applicants at child care centers reveal? 102-81.30 Section 102-81.30 Public Contracts and Property... PROPERTY 81-SECURITY Security § 102-81.30 What information must job applicants at child care centers reveal... on the job application. Employment at a child care facility means any position that involves...

  16. Information Gathering Revealed within the Social Network of Line-Managers.

    ERIC Educational Resources Information Center

    Mackenzie, Maureen L.

    2003-01-01

    Results of this study revealed that relationship, more than knowledge, may be the reason a manager is sought as an information source within a business environment. Social network mapping was used to capture a more intimate view of the information relationships within a business environment. Content analysis was used to analyze the data and to…

  17. Empirical Complexities in the Genetic Foundations of Lethal Mutagenesis

    PubMed Central

    Bull, James J.; Joyce, Paul; Gladstone, Eric; Molineux, Ian J.

    2013-01-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias—mutagenesis of virions—but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it. PMID:23934886

  18. Time course of information representation of macaque AIP neurons in hand manipulation task revealed by information analysis.

    PubMed

    Sakaguchi, Yutaka; Ishida, Fumihiko; Shimizu, Takashi; Murata, Akira

    2010-12-01

    We used mutual information analysis of neuronal activity in the macaque anterior intraparietal area (AIP) to examine information processing during a hand manipulation task. The task was to reach-to-grasp a three-dimensional (3D) object after presentation of a go signal. Mutual information was calculated between the spike counts of individual neurons in 50-ms-wide time bins and six unique shape classifications or 15 one-versus-one classifications of these shapes. The spatiotemporal distribution of mutual information was visualized as a two-dimensional image ("information map") to better observe global profiles of information representation. In addition, a nonnegative matrix factorization technique was applied for extracting its structure. Our major finding was that the time course of mutual information differed significantly according to different classes of task-related neurons. This strongly suggests that different classes of neurons were engaged in different information processing stages in executing the hand manipulation task. On the other hand, our analysis revealed the heterogeneous nature of information representation of AIP neurons. For example, "information latency" (or information onset) varied among individual neurons even in the same neuron class and the same shape classification. Further, some neurons changed "information preference" (i.e., shape classification with the largest amount of information) across different task periods. These suggest that neurons encode different information in the different task periods. Taking the present result together with previous findings, we used a Gantt chart to propose a hypothetical scheme of the dynamic interactions between different types of AIP neurons.

  19. Money makes you reveal more: consequences of monetary cues on preferential disclosure of personal information.

    PubMed

    Mukherjee, Sumitava; Manjaly, Jaison A; Nargundkar, Maithilee

    2013-01-01

    With continuous growth in information aggregation and dissemination, studies on privacy preferences are important to understand what makes people reveal information about them. Previous studies have demonstrated that short-term gains and possible monetary rewards make people risk disclosing information. Given the malleability of privacy preferences and the ubiquitous monetary cues in daily lives, we measured the contextual effect of reminding people about money on their privacy disclosure preferences. In experiment 1, we found that priming money increased willingness to disclose their personal information that could be shared with an online shopping website. Beyond stated willingness, experiment 2 tested whether priming money increases propensity for actually giving out personal information. Across both experiments, we found that priming money increases both the reported willingness and the actual disclosure of personal information. Our results imply that not only do short-term rewards make people trade-off personal security and privacy, but also mere exposure to money increases self-disclosure.

  20. 41 CFR 102-81.30 - What information must job applicants at child care centers reveal?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... job applicants at child care centers reveal? 102-81.30 Section 102-81.30 Public Contracts and Property Management Federal Property Management Regulations System (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 81-SECURITY Security § 102-81.30 What information must job applicants at child care centers...

  1. 41 CFR 102-81.30 - What information must job applicants at child care centers reveal?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false What information must job applicants at child care centers reveal? 102-81.30 Section 102-81.30 Public Contracts and Property Management Federal Property Management Regulations System (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 81-SECURITY Security §...

  2. In vitro models of mutagenesis.

    PubMed

    Strauss, B S; Larson, K; Sagher, D; Rabkin, S; Shenkar, R; Sahm, J

    1985-01-01

    The bypass of lesions in DNA with insertion of nucleotides opposite damaged bases has been studied as a model for mutagenesis in an in vitro system. Lesions introduced by dimethyl sulfate at adenines and by ultraviolet light at pyrimidine dimers act as termination sites on both double- and single-stranded DNA templates. Base selection opposite noninformational lesions is, in part, a property of the polymerases: different polymerases have different selectivities although all polymerases tested seem to prefer purines. The ability to insert "incorrect" bases is determined in part by the sequence 5' to the lesion on the template strand. The hypothesis that damaged purines tend to result in transversions can be applied to published data on activation of the c-ras oncogene.

  3. Combining complexity measures of EEG data: multiplying measures reveal previously hidden information.

    PubMed

    Burns, Thomas; Rajan, Ramesh

    2015-01-01

    Many studies have noted significant differences among human electroencephalograph (EEG) results when participants or patients are exposed to different stimuli, undertaking different tasks, or being affected by conditions such as epilepsy or Alzheimer's disease. Such studies often use only one or two measures of complexity and do not regularly justify their choice of measure beyond the fact that it has been used in previous studies. If more measures were added to such studies, however, more complete information might be found about these reported differences. Such information might be useful in confirming the existence or extent of such differences, or in understanding their physiological bases. In this study we analysed publically-available EEG data using a range of complexity measures to determine how well the measures correlated with one another. The complexity measures did not all significantly correlate, suggesting that different measures were measuring unique features of the EEG signals and thus revealing information which other measures were unable to detect. Therefore, the results from this analysis suggests that combinations of complexity measures reveal unique information which is in addition to the information captured by other measures of complexity in EEG data. For this reason, researchers using individual complexity measures for EEG data should consider using combinations of measures to more completely account for any differences they observe and to ensure the robustness of any relationships identified.

  4. Combining complexity measures of EEG data: multiplying measures reveal previously hidden information

    PubMed Central

    Burns, Thomas; Rajan, Ramesh

    2015-01-01

    Many studies have noted significant differences among human electroencephalograph (EEG) results when participants or patients are exposed to different stimuli, undertaking different tasks, or being affected by conditions such as epilepsy or Alzheimer's disease. Such studies often use only one or two measures of complexity and do not regularly justify their choice of measure beyond the fact that it has been used in previous studies. If more measures were added to such studies, however, more complete information might be found about these reported differences. Such information might be useful in confirming the existence or extent of such differences, or in understanding their physiological bases. In this study we analysed publically-available EEG data using a range of complexity measures to determine how well the measures correlated with one another. The complexity measures did not all significantly correlate, suggesting that different measures were measuring unique features of the EEG signals and thus revealing information which other measures were unable to detect. Therefore, the results from this analysis suggests that combinations of complexity measures reveal unique information which is in addition to the information captured by other measures of complexity in EEG data. For this reason, researchers using individual complexity measures for EEG data should consider using combinations of measures to more completely account for any differences they observe and to ensure the robustness of any relationships identified. PMID:26594331

  5. What genomic sequence information has revealed about Vibrio ecology in the ocean--a review.

    PubMed

    Grimes, Darrell Jay; Johnson, Crystal N; Dillon, Kevin S; Flowers, Adrienne R; Noriea, Nicholas F; Berutti, Tracy

    2009-10-01

    To date, the genomes of eight Vibrio strains representing six species and three human pathogens have been fully sequenced and reported. This review compares genomic information revealed from these sequencing efforts and what we can infer about Vibrio biology and ecology from this and related genomic information. The focus of the review is on those attributes that allow the Vibrios to survive and even proliferate in their ocean habitats, which include seawater, plankton, invertebrates, fish, marine mammals, plants, man-made structures (surfaces), and particulate matter. Areas covered include general information about the eight genomes, each of which is distributed over two chromosomes; a discussion of expected and unusual genes found; attachment sites and mechanisms; utilization of particulate and dissolved organic matter; and conclusions.

  6. Visualising the invisible: a network approach to reveal the informal social side of student learning.

    PubMed

    Hommes, J; Rienties, B; de Grave, W; Bos, G; Schuwirth, L; Scherpbier, A

    2012-12-01

    World-wide, universities in health sciences have transformed their curriculum to include collaborative learning and facilitate the students' learning process. Interaction has been acknowledged to be the synergistic element in this learning context. However, students spend the majority of their time outside their classroom and interaction does not stop outside the classroom. Therefore we studied how informal social interaction influences student learning. Moreover, to explore what really matters in the students learning process, a model was tested how the generally known important constructs-prior performance, motivation and social integration-relate to informal social interaction and student learning. 301 undergraduate medical students participated in this cross-sectional quantitative study. Informal social interaction was assessed using self-reported surveys following the network approach. Students' individual motivation, social integration and prior performance were assessed by the Academic Motivation Scale, the College Adaption Questionnaire and students' GPA respectively. A factual knowledge test represented student' learning. All social networks were positively associated with student learning significantly: friendships (β = 0.11), providing information to other students (β = 0.16), receiving information from other students (β = 0.25). Structural equation modelling revealed a model in which social networks increased student learning (r = 0.43), followed by prior performance (r = 0.31). In contrast to prior literature, students' academic motivation and social integration were not associated with students' learning. Students' informal social interaction is strongly associated with students' learning. These findings underline the need to change our focus from the formal context (classroom) to the informal context to optimize student learning and deliver modern medics.

  7. Insertional mutagenesis and illegitimate recombination in mycobacteria.

    PubMed Central

    Kalpana, G V; Bloom, B R; Jacobs, W R

    1991-01-01

    Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG. Images PMID:2052623

  8. Hierarchical structure of the Sicilian goats revealed by Bayesian analyses of microsatellite information.

    PubMed

    Siwek, M; Finocchiaro, R; Curik, I; Portolano, B

    2011-02-01

    Genetic structure and relationship amongst the main goat populations in Sicily (Girgentana, Derivata di Siria, Maltese and Messinese) were analysed using information from 19 microsatellite markers genotyped on 173 individuals. A posterior Bayesian approach implemented in the program STRUCTURE revealed a hierarchical structure with two clusters at the first level (Girgentana vs. Messinese, Derivata di Siria and Maltese), explaining 4.8% of variation (amovaФ(ST) estimate). Seven clusters nested within these first two clusters (further differentiations of Girgentana, Derivata di Siria and Maltese), explaining 8.5% of variation (amovaФ(SC) estimate). The analyses and methods applied in this study indicate their power to detect subtle population structure.

  9. Maize-targeted mutagenesis: A knockout resource for maize.

    PubMed

    May, Bruce P; Liu, Hong; Vollbrecht, Erik; Senior, Lynn; Rabinowicz, Pablo D; Roh, Donna; Pan, Xiaokang; Stein, Lincoln; Freeling, Mike; Alexander, Danny; Martienssen, Rob

    2003-09-30

    We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

  10. Mutual information reveals multiple structural relaxation mechanisms in a model glass former

    PubMed Central

    Dunleavy, Andrew J.; Wiesner, Karoline; Yamamoto, Ryoichi; Royall, C. Patrick

    2015-01-01

    Among the key challenges to our understanding of solidification in the glass transition is that it is accompanied by little apparent change in structure. Recently, geometric motifs have been identified in glassy liquids, but a causal link between these motifs and solidification remains elusive. One ‘smoking gun’ for such a link would be identical scaling of structural and dynamic lengthscales on approaching the glass transition, but this is highly controversial. Here we introduce an information theoretic approach to determine correlations in displacement for particle relaxation encoded in the initial configuration of a glass-forming liquid. We uncover two populations of particles, one inclined to relax quickly, the other slowly. Each population is correlated with local density and geometric motifs. Our analysis further reveals a dynamic lengthscale similar to that associated with structural properties, which may resolve the discrepancy between structural and dynamic lengthscales. PMID:25608791

  11. Correlates of perceptual awareness in human primary auditory cortex revealed by an informational masking experiment.

    PubMed

    Wiegand, Katrin; Gutschalk, Alexander

    2012-05-15

    The presence of an auditory event may remain undetected in crowded environments, even when it is well above the sensory threshold. This effect, commonly known as informational masking, allows for isolating neural activity related to perceptual awareness, by comparing repetitions of the same physical stimulus where the target is either detected or not. Evidence from magnetoencephalography (MEG) suggests that auditory-cortex activity in the latency range 50-250 ms is closely coupled with perceptual awareness. Here, BOLD fMRI and MEG were combined to investigate at which stage in the auditory cortex neural correlates of conscious auditory perception can be observed. Participants were asked to indicate the perception of a regularly repeating target tone, embedded within a random multi-tone masking background. Results revealed widespread activation within the auditory cortex for detected target tones, which was delayed but otherwise similar to the activation of an unmasked control stimulus. The contrast of detected versus undetected targets revealed activity confined to medial Heschl's gyrus, where the primary auditory cortex is located. These results suggest that activity related to conscious perception involves the primary auditory cortex and is not restricted to activity in secondary areas.

  12. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    PubMed

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  13. Genetic Code Evolution Reveals the Neutral Emergence of Mutational Robustness, and Information as an Evolutionary Constraint

    PubMed Central

    Massey, Steven E.

    2015-01-01

    The standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of “neutral emergence”. The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these “pseudaptations”, and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an “unfreezing” of the codon – amino acid mapping that defines the genetic code, consistent with Crick’s Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content

  14. Within- and Cross-Participant Classifiers Reveal Different Neural Coding of Information

    PubMed Central

    Clithero, John A.; Smith, David V.; Carter, R. McKell; Huettel, Scott A.

    2010-01-01

    Analyzing distributed patterns of brain activation using multivariate pattern analysis (MVPA) has become a popular approach for using functional magnetic resonance imaging (fMRI) data to predict mental states. While the majority of studies currently build separate classifiers for each participant in the sample, in principle a single classifier can be derived from and tested on data from all participants. These two approaches, within- and cross-participant classification, rely on potentially different sources of variability and thus may provide distinct information about brain function. Here, we used both approaches to identify brain regions that contain information about passively-received monetary rewards (i.e., images of currency that influenced participant payment) and social rewards (i.e., images of human faces). Our within-participant analyses implicated regions in the ventral visual processing stream – including fusiform gyrus and primary visual cortex – and ventromedial prefrontal cortex (VMPFC). Two key results indicate these regions may contain statistically discriminable patterns that contain different informational representations. First, cross-participant analyses implicated additional brain regions, including striatum and anterior insula. The cross-participant analyses also revealed systematic changes in predictive power across brain regions, with the pattern of change consistent with the functional properties of regions. Second, individual differences in classifier performance in VMPFC were related to individual differences in preferences between our two reward modalities. We interpret these results as reflecting a distinction between patterns reflecting participant-specific functional organization and those indicating aspects of brain organization that generalize across individuals. PMID:20347995

  15. Intracranial recordings reveal transient response dynamics during information maintenance in human cerebral cortex.

    PubMed

    Noy, Niv; Bickel, Stephan; Zion-Golumbic, Elana; Harel, Michal; Golan, Tal; Davidesco, Ido; Schevon, Catherine A; McKhann, Guy M; Goodman, Robert R; Schroeder, Charles E; Mehta, Ashesh D; Malach, Rafael

    2015-10-01

    Despite an extensive body of work, it is still not clear how short term maintenance of information is implemented in the human brain. Most prior research has focused on "working memory"-typically involving the storage of a number of items, requiring the use of a phonological loop and focused attention during the delay period between encoding and retrieval. These studies largely support a model of enhanced activity in the delay interval as the central mechanism underlying working memory. However, multi-item working memory constitutes only a subset of storage phenomena that may occur during daily life. A common task in naturalistic situations is short term memory of a single item-for example, blindly reaching to a previously placed cup of coffee. Little is known about such single-item, effortless, storage in the human brain. Here, we examined the dynamics of brain responses during a single-item maintenance task, using intracranial recordings implanted for clinical purpose in patients (ECoG). Our results reveal that active electrodes were dominated by transient short latency visual and motor responses, reflected in broadband high frequency power increases in occipito-temporal, frontal, and parietal cortex. Only a very small set of electrodes showed activity during the early part of the delay period. Interestingly, no cortical site displayed a significant activation lasting to the response time. These results suggest that single item encoding is characterized by transient high frequency ECoG responses, while the maintenance of information during the delay period may be mediated by mechanisms necessitating only low-levels of neuronal activations.

  16. Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

    ERIC Educational Resources Information Center

    Hartberg, Yasha

    2006-01-01

    By shifting pedagogical goals from obtaining successful mutations to teaching students critical troubleshooting skills, it has been possible to introduce site-directed mutagenesis into an undergraduate teaching laboratory. Described in this study is an inexpensive laboratory exercise in which students follow a slightly modified version of…

  17. CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS

    EPA Science Inventory

    CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS
    Michael D. Waters
    US Environmental Protection Agency, MD-51A, Research Triangle Park, NC 27711 USA

    Our rapidly growing understanding of the structure of the human genome is forming the basis for numerous new...

  18. The evolutionary function of conscious information processing is revealed by its task-dependency in the olfactory system.

    PubMed

    Keller, Andreas

    2014-01-01

    Although many responses to odorous stimuli are mediated without olfactory information being consciously processed, some olfactory behaviors require conscious information processing. I will here contrast situations in which olfactory information is processed consciously to situations in which it is processed non-consciously. This contrastive analysis reveals that conscious information processing is required when an organism is faced with tasks in which there are many behavioral options available. I therefore propose that it is the evolutionary function of conscious information processing to guide behaviors in situations in which the organism has to choose between many possible responses.

  19. The utility of transposon mutagenesis for cancer studies in the era of genome editing.

    PubMed

    DeNicola, Gina M; Karreth, Florian A; Adams, David J; Wong, Chi C

    2015-10-19

    The use of transposons as insertional mutagens to identify cancer genes in mice has generated a wealth of information over the past decade. Here, we discuss recent major advances in transposon-mediated insertional mutagenesis screens and compare this technology with other screening strategies.

  20. New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis

    PubMed Central

    2013-01-01

    Background In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions INGenomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. Results In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish

  1. Novel Random Mutagenesis Method for Directed Evolution.

    PubMed

    Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

    2017-01-01

    Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

  2. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  3. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.

    PubMed

    Fukuda, Masatora; Umeno, Hiromitsu; Nose, Kanako; Nishitarumizu, Azusa; Noguchi, Ryoma; Nakagawa, Hiroyuki

    2017-02-02

    As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.

  4. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing

    PubMed Central

    Fukuda, Masatora; Umeno, Hiromitsu; Nose, Kanako; Nishitarumizu, Azusa; Noguchi, Ryoma; Nakagawa, Hiroyuki

    2017-01-01

    As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method. PMID:28148949

  5. Risk and Ambiguity in Information Seeking: Eye Gaze Patterns Reveal Contextual Behavior in Dealing with Uncertainty

    PubMed Central

    Wittek, Peter; Liu, Ying-Hsang; Darányi, Sándor; Gedeon, Tom; Lim, Ik Soo

    2016-01-01

    Information foraging connects optimal foraging theory in ecology with how humans search for information. The theory suggests that, following an information scent, the information seeker must optimize the tradeoff between exploration by repeated steps in the search space vs. exploitation, using the resources encountered. We conjecture that this tradeoff characterizes how a user deals with uncertainty and its two aspects, risk and ambiguity in economic theory. Risk is related to the perceived quality of the actually visited patch of information, and can be reduced by exploiting and understanding the patch to a better extent. Ambiguity, on the other hand, is the opportunity cost of having higher quality patches elsewhere in the search space. The aforementioned tradeoff depends on many attributes, including traits of the user: at the two extreme ends of the spectrum, analytic and wholistic searchers employ entirely different strategies. The former type focuses on exploitation first, interspersed with bouts of exploration, whereas the latter type prefers to explore the search space first and consume later. Our findings from an eye-tracking study of experts' interactions with novel search interfaces in the biomedical domain suggest that user traits of cognitive styles and perceived search task difficulty are significantly correlated with eye gaze and search behavior. We also demonstrate that perceived risk shifts the balance between exploration and exploitation in either type of users, tilting it against vs. in favor of ambiguity minimization. Since the pattern of behavior in information foraging is quintessentially sequential, risk and ambiguity minimization cannot happen simultaneously, leading to a fundamental limit on how good such a tradeoff can be. This in turn connects information seeking with the emergent field of quantum decision theory. PMID:27909418

  6. Risk and Ambiguity in Information Seeking: Eye Gaze Patterns Reveal Contextual Behavior in Dealing with Uncertainty.

    PubMed

    Wittek, Peter; Liu, Ying-Hsang; Darányi, Sándor; Gedeon, Tom; Lim, Ik Soo

    2016-01-01

    Information foraging connects optimal foraging theory in ecology with how humans search for information. The theory suggests that, following an information scent, the information seeker must optimize the tradeoff between exploration by repeated steps in the search space vs. exploitation, using the resources encountered. We conjecture that this tradeoff characterizes how a user deals with uncertainty and its two aspects, risk and ambiguity in economic theory. Risk is related to the perceived quality of the actually visited patch of information, and can be reduced by exploiting and understanding the patch to a better extent. Ambiguity, on the other hand, is the opportunity cost of having higher quality patches elsewhere in the search space. The aforementioned tradeoff depends on many attributes, including traits of the user: at the two extreme ends of the spectrum, analytic and wholistic searchers employ entirely different strategies. The former type focuses on exploitation first, interspersed with bouts of exploration, whereas the latter type prefers to explore the search space first and consume later. Our findings from an eye-tracking study of experts' interactions with novel search interfaces in the biomedical domain suggest that user traits of cognitive styles and perceived search task difficulty are significantly correlated with eye gaze and search behavior. We also demonstrate that perceived risk shifts the balance between exploration and exploitation in either type of users, tilting it against vs. in favor of ambiguity minimization. Since the pattern of behavior in information foraging is quintessentially sequential, risk and ambiguity minimization cannot happen simultaneously, leading to a fundamental limit on how good such a tradeoff can be. This in turn connects information seeking with the emergent field of quantum decision theory.

  7. Network information analysis reveals risk perception transmission in a behaviour-influenza dynamics system.

    PubMed

    Liao, C-M; You, S-H; Cheng, Y-H

    2015-01-01

    Influenza poses a significant public health burden worldwide. Understanding how and to what extent people would change their behaviour in response to influenza outbreaks is critical for formulating public health policies. We incorporated the information-theoretic framework into a behaviour-influenza (BI) transmission dynamics system in order to understand the effects of individual behavioural change on influenza epidemics. We showed that information transmission of risk perception played a crucial role in the spread of health-seeking behaviour throughout influenza epidemics. Here a network BI model provides a new approach for understanding the risk perception spread and human behavioural change during disease outbreaks. Our study allows simultaneous consideration of epidemiological, psychological, and social factors as predictors of individual perception rates in behaviour-disease transmission systems. We suggest that a monitoring system with precise information on risk perception should be constructed to effectively promote health behaviours in preparation for emerging disease outbreaks.

  8. A novel dynamics combination model reveals the hidden information of community structure

    NASA Astrophysics Data System (ADS)

    Li, Hui-Jia; Li, Huiying; Jia, Chuanliang

    2015-09-01

    The analysis of the dynamic details of community structure is an important question for scientists from many fields. In this paper, we propose a novel Markov-Potts framework to uncover the optimal community structures and their stabilities across multiple timescales. Specifically, we model the Potts dynamics to detect community structure by a Markov process, which has a clear mathematical explanation. Then the local uniform behavior of spin values revealed by our model is shown that can naturally reveal the stability of hierarchical community structure across multiple timescales. To prove the validity, phase transition of stochastic dynamic system is used to indicate that the stability of community structure we proposed is able to describe the significance of community structure based on eigengap theory. Finally, we test our framework on some example networks and find it does not have resolute limitation problem at all. Results have shown the model we proposed is able to uncover hierarchical structure in different scales effectively and efficiently.

  9. Multivoxel pattern analysis reveals 3D place information in the human hippocampus.

    PubMed

    Kim, Misun; Jeffery, Kate J; Maguire, Eleanor A

    2017-03-20

    The spatial world is three-dimensional (3D), and humans and other animals move both horizontally and vertically within it. Extant neuroscientific studies have typically investigated spatial navigation on a horizontal two-dimensional plane, leaving much unknown about how 3D spatial information is represented in the brain. Specifically, horizontal and vertical information may be encoded in the same or different neural structures with equal or unequal sensitivity. Here, we investigated these possibilities using functional MRI (fMRI) while participants were passively moved within a 3D lattice structure as if riding a rollercoaster. Multivoxel pattern analysis was used to test for the existence of information relating to where and in which direction participants were heading in this virtual environment. Behaviorally, participants had similarly accurate memory for vertical and horizontal locations, and the right anterior hippocampus expressed place information that was sensitive to changes along both horizontal and vertical axes. This is suggestive of isotropic 3D place encoding. By contrast, participants indicated their heading direction faster and more accurately when they were heading in a tilted-up or tilted-down direction. This direction information was expressed in the right retrosplenial cortex and posterior hippocampus, and was only sensitive to vertical pitch, which could reflect the importance of the vertical (gravity) axis as a reference frame. Overall, our findings extend previous knowledge of how we represent the spatial world and navigate within it, by taking into account the important third dimension.SIGNIFICANCE STATEMENTThe spatial world is three-dimensional (3D) -- we can move horizontally across surfaces, but also vertically, going up slopes or stairs. Little is known about how the brain supports representations of 3D space. A key question is whether or not horizontal and vertical information is equally well represented. Here we measured functional MRI

  10. AS52/GPT Mammalian Mutagenesis Assay

    DTIC Science & Technology

    1996-05-10

    dimethylnitrosamine (DMN) at 50 and 100 f.J.g/rnl was used as a 3 TLS Project Nn. A0ŗ-003: AS52/GPT Mammalian Mutagenesis Assay promutagen that requires metabolic...Chemical Source Lot No. air Air Products N/A calcium chloride Sigma 84F-0723 d imeth y !sulfoxide Fisher 933274 dimethylnitrosamine Sigma 82B0365...methanesulfonate (EMS) at 150 and 300 J.i-g/ml is used as a direct-acting mutagen for the nonactivated portion, and dimethylnitrosamine (DMN) at 150 and 300

  11. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, Frank A.

    1982-01-01

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  12. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, F.A.

    1980-12-12

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  13. New insights into behaviour using mouse ENU mutagenesis.

    PubMed

    Oliver, Peter L; Davies, Kay E

    2012-10-15

    Identifying genes involved in behavioural disorders in man is a challenge as the cause is often multigenic and the phenotype is modulated by environmental cues. Mouse mutants are a valuable tool for identifying novel pathways underlying specific neurological phenotypes and exploring the influence both genetic and non-genetic factors. Many human variants causing behavioural disorders are not gene deletions but changes in levels of expression or activity of a gene product; consequently, large-scale mouse ENU mutagenesis has the advantage over the study of null mutants in that it generates a range of point mutations that frequently mirror the subtlety and heterogeneity of human genetic lesions. ENU mutants have provided novel and clinically relevant functional information on genes that influence many aspects of mammalian behaviour, from neuropsychiatric endophenotypes to circadian rhythms. This review will highlight some of the most important findings that have been made using this method in several key areas of neurological disease research.

  14. Dynamic information processing states revealed through neurocognitive models of object semantics

    PubMed Central

    Clarke, Alex

    2015-01-01

    Recognising objects relies on highly dynamic, interactive brain networks to process multiple aspects of object information. To fully understand how different forms of information about objects are represented and processed in the brain requires a neurocognitive account of visual object recognition that combines a detailed cognitive model of semantic knowledge with a neurobiological model of visual object processing. Here we ask how specific cognitive factors are instantiated in our mental processes and how they dynamically evolve over time. We suggest that coarse semantic information, based on generic shared semantic knowledge, is rapidly extracted from visual inputs and is sufficient to drive rapid category decisions. Subsequent recurrent neural activity between the anterior temporal lobe and posterior fusiform supports the formation of object-specific semantic representations – a conjunctive process primarily driven by the perirhinal cortex. These object-specific representations require the integration of shared and distinguishing object properties and support the unique recognition of objects. We conclude that a valuable way of understanding the cognitive activity of the brain is though testing the relationship between specific cognitive measures and dynamic neural activity. This kind of approach allows us to move towards uncovering the information processing states of the brain and how they evolve over time. PMID:25745632

  15. Immediate relativity: EEG reveals early engagement of comparison in social information processing.

    PubMed

    Ohmann, Katharina; Stahl, Jutta; Mussweiler, Thomas; Kedia, Gayannée

    2016-11-01

    A wide array of social decisions relies on social comparisons. As such, these decisions require fast access to relative information. Therefore, we expect that signatures of the comparative process should be observable in electrophysiological components at an early stage of information processing. However, to date, little is known about the neural time course of social target comparisons. Therefore, we tested this hypothesis in 2 electroencephalography (EEG) studies using a social distance effect paradigm. The distance effect capitalizes on the fact that stimuli close on a certain dimension take longer to compare than stimuli clearly differing on this dimension. Here, we manipulated the distance of face characteristics regarding their levels of attractiveness (Study 1) and trustworthiness (Study 2), 2 essential social dimensions. In both studies, size comparisons served as a nonsocial control condition. In Study 1, distance related effects were apparent 170 ms (vertex positive potential, VPP) and 200 ms (N2) after stimulus onset for attractiveness comparisons. In Study 2, trustworthiness comparisons took effect already after 100 ms (N1) and likewise carried over to an event-related N2. Remarkably, we observed a similar temporal pattern for social (attractiveness, trustworthiness) and nonsocial (size) dimensions. These results speak in favor of an early encoding of comparative information and emphasize the primary role of comparison in social information processing. (PsycINFO Database Record

  16. Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation

    PubMed Central

    Guschinskaya, Natalia; Brunel, Romain; Tourte, Maxime; Lipscomb, Gina L.; Adams, Michael W. W.; Oger, Philippe; Charpentier, Xavier

    2016-01-01

    Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes. PMID:27824140

  17. Trans-saccadic repetition priming: ERPs reveal on-line integration of information across words.

    PubMed

    Grainger, Jonathan; Midgley, Katherine J; Holcomb, Phillip J

    2016-01-08

    We used a trans-saccadic priming paradigm combined with ERP recordings to track the time-course of integration of information across a prime word briefly presented at fixation and a subsequent target word presented 4 degrees to the right of fixation. Trans-saccadic repetition priming effects (Experiments 1 and 2) were compared with priming effects obtained with centrally located targets (Experiment 3). In Experiment 2, target stimuli were preceded by a 100ms forward mask at the target location, hence allowing an attention shift to the target location prior to target onset. Compared with centrally located targets, repetition priming effects were found to onset later in Experiment 2 and even later in Experiment 1, and the growth of priming effects was slower in both Experiments 1 and 2 compared with Experiment 3. The results demonstrate integration of information across spatially distinct primes and targets, with the time-course of trans-saccadic priming being determined by the speed with which attention can be allocated to peripheral targets plus the quality of information available in peripheral vision prior to fixation of target stimuli.

  18. Digital Health: Tracking Physiomes and Activity Using Wearable Biosensors Reveals Useful Health-Related Information

    PubMed Central

    Zhou, Gao; Zhou, Wenyu; Schüssler-Fiorenza Rose, Sophia Miryam; Perelman, Dalia; Colbert, Elizabeth; Runge, Ryan; Rego, Shannon; Sonecha, Ria; Datta, Somalee; McLaughlin, Tracey; Snyder, Michael P.

    2017-01-01

    A new wave of portable biosensors allows frequent measurement of health-related physiology. We investigated the use of these devices to monitor human physiological changes during various activities and their role in managing health and diagnosing and analyzing disease. By recording over 250,000 daily measurements for up to 43 individuals, we found personalized circadian differences in physiological parameters, replicating previous physiological findings. Interestingly, we found striking changes in particular environments, such as airline flights (decreased peripheral capillary oxygen saturation [SpO2] and increased radiation exposure). These events are associated with physiological macro-phenotypes such as fatigue, providing a strong association between reduced pressure/oxygen and fatigue on high-altitude flights. Importantly, we combined biosensor information with frequent medical measurements and made two important observations: First, wearable devices were useful in identification of early signs of Lyme disease and inflammatory responses; we used this information to develop a personalized, activity-based normalization framework to identify abnormal physiological signals from longitudinal data for facile disease detection. Second, wearables distinguish physiological differences between insulin-sensitive and -resistant individuals. Overall, these results indicate that portable biosensors provide useful information for monitoring personal activities and physiology and are likely to play an important role in managing health and enabling affordable health care access to groups traditionally limited by socioeconomic class or remote geography. PMID:28081144

  19. EEG-informed fMRI reveals spatiotemporal characteristics of perceptual decision making.

    PubMed

    Philiastides, Marios G; Sajda, Paul

    2007-11-28

    Single-unit and multiunit recordings in primates have already established that decision making involves at least two general stages of neural processing: representation of evidence from early sensory areas and accumulation of evidence to a decision threshold from decision-related regions. However, the relay of information from early sensory to decision areas, such that the accumulation process is instigated, is not well understood. Using a cued paradigm and single-trial analysis of electroencephalography (EEG), we previously reported on temporally specific components related to perceptual decision making. Here, we use information derived from our previous EEG recordings to inform the analysis of fMRI data collected for the same behavioral task to ascertain the cortical origins of each of these EEG components. We demonstrate that a cascade of events associated with perceptual decision making takes place in a highly distributed neural network. Of particular importance is an activation in the lateral occipital complex implicating perceptual persistence as a mechanism by which object decision making in the human brain is instigated.

  20. Digital Health: Tracking Physiomes and Activity Using Wearable Biosensors Reveals Useful Health-Related Information.

    PubMed

    Li, Xiao; Dunn, Jessilyn; Salins, Denis; Zhou, Gao; Zhou, Wenyu; Schüssler-Fiorenza Rose, Sophia Miryam; Perelman, Dalia; Colbert, Elizabeth; Runge, Ryan; Rego, Shannon; Sonecha, Ria; Datta, Somalee; McLaughlin, Tracey; Snyder, Michael P

    2017-01-01

    A new wave of portable biosensors allows frequent measurement of health-related physiology. We investigated the use of these devices to monitor human physiological changes during various activities and their role in managing health and diagnosing and analyzing disease. By recording over 250,000 daily measurements for up to 43 individuals, we found personalized circadian differences in physiological parameters, replicating previous physiological findings. Interestingly, we found striking changes in particular environments, such as airline flights (decreased peripheral capillary oxygen saturation [SpO2] and increased radiation exposure). These events are associated with physiological macro-phenotypes such as fatigue, providing a strong association between reduced pressure/oxygen and fatigue on high-altitude flights. Importantly, we combined biosensor information with frequent medical measurements and made two important observations: First, wearable devices were useful in identification of early signs of Lyme disease and inflammatory responses; we used this information to develop a personalized, activity-based normalization framework to identify abnormal physiological signals from longitudinal data for facile disease detection. Second, wearables distinguish physiological differences between insulin-sensitive and -resistant individuals. Overall, these results indicate that portable biosensors provide useful information for monitoring personal activities and physiology and are likely to play an important role in managing health and enabling affordable health care access to groups traditionally limited by socioeconomic class or remote geography.

  1. An efficient TALEN mutagenesis system in rice.

    PubMed

    Chen, Kunling; Shan, Qiwei; Gao, Caixia

    2014-08-15

    Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5 months.

  2. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  3. Environmental DNA reveals that rivers are conveyer belts of biodiversity information

    PubMed Central

    Deiner, Kristy; Fronhofer, Emanuel A.; Mächler, Elvira; Walser, Jean-Claude; Altermatt, Florian

    2016-01-01

    DNA sampled from the environment (eDNA) is a useful way to uncover biodiversity patterns. By combining a conceptual model and empirical data, we test whether eDNA transported in river networks can be used as an integrative way to assess eukaryotic biodiversity for broad spatial scales and across the land–water interface. Using an eDNA metabarcode approach, we detect 296 families of eukaryotes, spanning 19 phyla across the catchment of a river. We show for a subset of these families that eDNA samples overcome spatial autocorrelation biases associated with the classical community assessments by integrating biodiversity information over space. In addition, we demonstrate that many terrestrial species are detected; thus suggesting eDNA in river water also incorporates biodiversity information across terrestrial and aquatic biomes. Environmental DNA transported in river networks offers a novel and spatially integrated way to assess the total biodiversity for whole landscapes and will transform biodiversity data acquisition in ecology. PMID:27572523

  4. Using statistical reasoning performance to reveal information parsing preferences in the mind.

    PubMed

    Brase, Gary L

    2015-01-01

    Many cognitive tasks require the parsing of information into smaller, discrete units in order to enable effective information processing. This parsing can, broadly speaking, be done along either situationally ad hoc dimensions or done preferentially along ecologically and evolutionarily relevant dimensions. The present research systematically evaluates these two possibilities within a statistical reasoning context. While replicating results that appear to support the partition-edit-count hypothesis (that item parsing is equipotential, based on subtle linguistic cues), this result was found to be in large part due to confounds in the nature of the tasks rather than the partitioning manipulations (Experiment 1). Additionally, a frequency presentation of the same task not only eliminated the earlier confounds but also improved performance directly and as predicted by the alternative hypothesis (Experiment 2). Attempts to reintroduce a biasing partition frame (Experiment 3) and a process study of participants' task representation (Experiment 4) also both failed to support the partition-edit-count hypothesis. These results favour an ecological rationality perspective and the associated frequency and individuation hypotheses regarding statistical reasoning (i.e., a privileged status for frequency representations to guide parsing of objects, events, and locations into easily countable units).

  5. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

    PubMed Central

    Canver, Matthew C.; Smith, Elenoe C.; Sher, Falak; Pinello, Luca; Sanjana, Neville E.; Shalem, Ophir; Chen, Diane D.; Schupp, Patrick G.; Vinjamur, Divya S.; Garcia, Sara P.; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Feng, Zhang; Orkin, Stuart H.; Bauer, Daniel E.

    2015-01-01

    Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements. PMID:26375006

  6. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

    PubMed

    Canver, Matthew C; Smith, Elenoe C; Sher, Falak; Pinello, Luca; Sanjana, Neville E; Shalem, Ophir; Chen, Diane D; Schupp, Patrick G; Vinjamur, Divya S; Garcia, Sara P; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Zhang, Feng; Orkin, Stuart H; Bauer, Daniel E

    2015-11-12

    Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

  7. The Rational Adolescent: Strategic Information Processing during Decision Making Revealed by Eye Tracking

    PubMed Central

    Kwak, Youngbin; Payne, John W.; Cohen, Andrew L.; Huettel, Scott A.

    2015-01-01

    Adolescence is often viewed as a time of irrational, risky decision-making – despite adolescents' competence in other cognitive domains. In this study, we examined the strategies used by adolescents (N=30) and young adults (N=47) to resolve complex, multi-outcome economic gambles. Compared to adults, adolescents were more likely to make conservative, loss-minimizing choices consistent with economic models. Eye-tracking data showed that prior to decisions, adolescents acquired more information in a more thorough manner; that is, they engaged in a more analytic processing strategy indicative of trade-offs between decision variables. In contrast, young adults' decisions were more consistent with heuristics that simplified the decision problem, at the expense of analytic precision. Collectively, these results demonstrate a counter-intuitive developmental transition in economic decision making: adolescents' decisions are more consistent with rational-choice models, while young adults more readily engage task-appropriate heuristics. PMID:26388664

  8. Not all order memory is equal: Test demands reveal dissociations in memory for sequence information.

    PubMed

    Jonker, Tanya R; MacLeod, Colin M

    2017-02-01

    Remembering the order of a sequence of events is a fundamental feature of episodic memory. Indeed, a number of formal models represent temporal context as part of the memory system, and memory for order has been researched extensively. Yet, the nature of the code(s) underlying sequence memory is still relatively unknown. Across 4 experiments that manipulated encoding task, we found evidence for 3 dissociable facets of order memory. Experiment 1 introduced a test requiring a judgment of which of 2 alternatives had immediately followed a word during encoding. This measure revealed better retention of interitem associations following relational encoding (silent reading) than relatively item-specific encoding (judging referent size), a pattern consistent with that observed in previous research using order reconstruction tests. In sharp contrast, Experiment 2 demonstrated the reverse pattern: Memory for the studied order of 2 sequentially presented items was actually better following item-specific encoding than following relational encoding. Experiment 3 reproduced this dissociation in a single experiment using both tests. Experiment 4 extended these findings by further dissociating the roles of relational encoding and item strength in the 2 tests. Taken together, these results indicate that memory for event sequence is influenced by (a) interitem associations, (b) the emphasized directionality of an association, and (c) an item's strength independent of other items. Memory for order is more complicated than has been portrayed in theories of memory and its nuances should be carefully considered when designing tests and models of temporal and relational memory. (PsycINFO Database Record

  9. Supra-additive contribution of shape and surface information to individual face discrimination as revealed by fast periodic visual stimulation.

    PubMed

    Dzhelyova, Milena; Rossion, Bruno

    2014-12-24

    Face perception depends on two main sources of information--shape and surface cues. Behavioral studies suggest that both of them contribute roughly equally to discrimination of individual faces, with only a small advantage provided by their combination. However, it is difficult to quantify the respective contribution of each source of information to the visual representation of individual faces with explicit behavioral measures. To address this issue, facial morphs were created that varied in shape only, surface only, or both. Electrocephalogram (EEG) were recorded from 10 participants during visual stimulation at a fast periodic rate, in which the same face was presented four times consecutively and the fifth face (the oddball) varied along one of the morphed dimensions. Individual face discrimination was indexed by the periodic EEG response at the oddball rate (e.g., 5.88 Hz/5 = 1.18 Hz). While shape information was discriminated mainly at right occipitotemporal electrode sites, surface information was coded more bilaterally and provided a larger response overall. Most importantly, shape and surface changes alone were associated with much weaker responses than when both sources of information were combined in the stimulus, revealing a supra-additive effect. These observations suggest that the two kinds of information combine nonlinearly to provide a full individual face representation, face identity being more than the sum of the contribution of shape and surface cues.

  10. New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis

    PubMed Central

    Wilson, Adam C.; Perego, Marta; Hoch, James A.

    2007-01-01

    Two new transposon delivery vector systems utilizing Mariner and mini-Tn10 transposons have been developed for in vivo insertional mutagenesis in Bacillus anthracis and other compatible Gram-positive species. The utility of both systems was directly demonstrated through the mutagenesis of a widely used B. anthracis strain. PMID:17931726

  11. Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening

    PubMed Central

    2011-01-01

    metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening. Conclusions Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit. PMID:22047180

  12. Shape information mediating basic- and subordinate-level object recognition revealed by analyses of eye movements.

    PubMed

    Davitt, Lina I; Cristino, Filipe; Wong, Alan C-N; Leek, E Charles

    2014-04-01

    This study examines the kinds of shape features that mediate basic- and subordinate-level object recognition. Observers were trained to categorize sets of novel objects at either a basic (between-families) or subordinate (within-family) level of classification. We analyzed the spatial distributions of fixations and compared them to model distributions of different curvature polarity (regions of convex or concave bounding contour), as well as internal part boundaries. The results showed a robust preference for fixation at part boundaries and for concave over convex regions of bounding contour, during both basic- and subordinate-level classification. In contrast, mean saccade amplitudes were shorter during basic- than subordinate-level classification. These findings challenge models of recognition that do not posit any special functional status to part boundaries or curvature polarity. We argue that both basic- and subordinate-level classification are mediated by object representations. These representations make explicit internal part boundaries, and distinguish concave and convex regions of bounding contour. The classification task constrains how shape information in these representations is used, consistent with the hypothesis that both parts-based, and image-based, operations support object recognition in human vision.

  13. Conditional gene-trap mutagenesis in zebrafish.

    PubMed

    Maddison, Lisette A; Li, Mingyu; Chen, Wenbiao

    2014-01-01

    Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.

  14. Mutagenesis assays of human amniotic fluid

    SciTech Connect

    Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.

    1985-01-01

    Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.

  15. From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

    PubMed Central

    Wright, Tom H.; Vallée, M. Robert J.

    2016-01-01

    Abstract Site‐directed (gene) mutagenesis has been the most useful method available for the conversion of one amino acid residue of a given protein into another. Until relatively recently, this strategy was limited to the twenty standard amino acids. The ongoing maturation of stop codon suppression and related technologies for unnatural amino acid incorporation has greatly expanded access to nonstandard amino acids by expanding the scope of the translational apparatus. However, the necessity for translation of genetic changes restricts the diversity of residues that may be incorporated. Herein we highlight an alternative approach, termed post‐expression mutagenesis, which operates at the level of the very functional biomolecules themselves. Using the lens of retrosynthesis, we highlight prospects for new strategies in protein modification, alteration, and construction which will enable protein science to move beyond the constraints of the “translational filter” and lead to a true synthetic biology. PMID:27119221

  16. The Effects of Revealed Information on Catastrophe Loss Projection Models' Characterization of Risk: Damage Vulnerability Evidence from Florida.

    PubMed

    Karl, J Bradley; Medders, Lorilee A; Maroney, Patrick F

    2016-06-01

    We examine whether the risk characterization estimated by catastrophic loss projection models is sensitive to the revelation of new information regarding risk type. We use commercial loss projection models from two widely employed modeling firms to estimate the expected hurricane losses of Florida Atlantic University's building stock, both including and excluding secondary information regarding hurricane mitigation features that influence damage vulnerability. We then compare the results of the models without and with this revealed information and find that the revelation of additional, secondary information influences modeled losses for the windstorm-exposed university building stock, primarily evidenced by meaningful percent differences in the loss exceedance output indicated after secondary modifiers are incorporated in the analysis. Secondary risk characteristics for the data set studied appear to have substantially greater impact on probable maximum loss estimates than on average annual loss estimates. While it may be intuitively expected for catastrophe models to indicate that secondary risk characteristics hold value for reducing modeled losses, the finding that the primary value of secondary risk characteristics is in reduction of losses in the "tail" (low probability, high severity) events is less intuitive, and therefore especially interesting. Further, we address the benefit-cost tradeoffs that commercial entities must consider when deciding whether to undergo the data collection necessary to include secondary information in modeling. Although we assert the long-term benefit-cost tradeoff is positive for virtually every entity, we acknowledge short-term disincentives to such an effort.

  17. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis

    PubMed Central

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J. Pablo; Lopez, Bernard S.

    2016-01-01

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses. PMID:27406380

  18. Heat shock and herpes virus: enhanced reactivation without untargeted mutagenesis

    SciTech Connect

    Lytle, C.D.; Carney, P.G.

    1988-01-01

    Enhanced reactivation of Ultraviolet-irradiated virus has been reported to occur in heat-shocked host cells. Since enhanced virus reactivation is often accompanied by untargeted mutagenesis, we investigated whether such mutagenesis would occur for herpes simplex virus (HSV) in CV-1 monkey kidney cells subjected to heat shock. In addition to expressing enhanced reactivation, the treated cells were transiently more susceptible to infection by unirradiated HSV. No mutagenesis of unirradiated HSV was found whether infection occurred at the time of increased susceptibility to infection or during expression of enhanced viral reactivation.

  19. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

    PubMed

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2014-01-01

    The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18-47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.

  20. Hepatic cirrhosis and recovery as reflected by Raman spectroscopy: information revealed by statistical analysis might lead to a prognostic biomarker.

    PubMed

    Galler, Kerstin; Fröhlich, Esther; Kortgen, Andreas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-11-01

    Hepatic cirrhosis is a severe chronic disease of the liver accompanied by massive changes in the physiology of the cells constituting the hepatic tissue. Success or failing of a therapeutic effort is difficult to recognize because of its late manifestation in the tissue morphology. In this study, the complex course of hepatic cirrhosis and its regression is followed in a rodent carbon tetrachloride model. Raman spectroscopy, which senses molecular vibrations and reflects the molecular composition of a sample, was applied to gain label- and destruction-free insights into the process of cirrhosis and to conclude on the hepatic disease state on the cellular level. Hematoxylin and eosin staining and immunofluorescence labeling were used to obtain complementary information. False color images derived from maps of Raman spectra by spectral unmixing revealed individual nucleus positions giving structural information. Spectral data unraveled chemical changes associated with liver damage on the cellular level. Upon carbon tetrachloride treatment, a higher lipid content and the presence of catabolic products indicated cirrhosis in tissue samples. Furthermore, 77 % of the Raman spectra recorded from treated rats were classified as diseased, whereas 96 % of the Raman spectra recorded from untreated rats were classified as healthy. Importantly, samples from rats that experienced a recovery period revealed a chemical composition highly similar to the ones from healthy rats while morphologically clear signs of tissue damage were still obvious. Eighty-three percent of their Raman spectra were classified as healthy. The vibrational fingerprint of tissue provides characteristic information that might serve as prognostic biomarker. Graphical Abstract Images of hematoxylin and eosin stained tissue slices together with an average Raman spectrum belonging to healthy and cirrhotic rat liver, respectively, visualize morphological and spectral differences between the two states of the

  1. Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology

    PubMed Central

    Lin, Tao; Troy, Erin B.; Hu, Linden T.; Gao, Lihui; Norris, Steven J.

    2014-01-01

    Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism. PMID:24904839

  2. Mutagenesis during plant responses to UVB radiation.

    PubMed

    Holá, M; Vágnerová, R; Angelis, K J

    2015-08-01

    We tested an idea that induced mutagenesis due to unrepaired DNA lesions, here the UV photoproducts, underlies the impact of UVB irradiation on plant phenotype. For this purpose we used protonemal culture of the moss Physcomitrella patens with 50% of apical cells, which mimics actively growing tissue, the most vulnerable stage for the induction of mutations. We measured the UVB mutation rate of various moss lines with defects in DNA repair (pplig4, ppku70, pprad50, ppmre11), and in selected clones resistant to 2-Fluoroadenine, which were mutated in the adenosine phosphotrasferase gene (APT), we analysed induced mutations by sequencing. In parallel we followed DNA break repair and removal of cyclobutane pyrimidine dimers with a half-life τ = 4 h 14 min determined by comet assay combined with UV dimer specific T4 endonuclease V. We show that UVB induces massive, sequence specific, error-prone bypass repair that is responsible for a high mutation rate owing to relatively slow, though error-free, removal of photoproducts by nucleotide excision repair (NER).

  3. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  4. A mutagenesis study of a catalytic antibody

    SciTech Connect

    Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.; Schultz, P.G. )

    1991-01-01

    The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and four Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.

  5. Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans

    PubMed Central

    Banh, Quyen; Arenskötter, Matthias; Steinbüchel, Alexander

    2005-01-01

    The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons. PMID:16151089

  6. Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis.

    PubMed Central

    Errampalli, D; Patton, D; Castle, L; Mickelson, L; Hansen, K; Schnall, J; Feldmann, K; Meinke, D

    1991-01-01

    T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis. PMID:12324593

  7. Symposium on molecular and cellular mechanisms of mutagenesis

    SciTech Connect

    Not Available

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents. (ERB)

  8. Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

    PubMed

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-10-21

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

  9. Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

    PubMed Central

    Caignard, Grégory; Eva, Megan M.; van Bruggen, Rebekah; Eveleigh, Robert; Bourque, Guillaume; Malo, Danielle; Gros, Philippe; Vidal, Silvia M.

    2014-01-01

    Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses. PMID:25268389

  10. Strong genetic admixture in the Altai at the Middle Bronze Age revealed by uniparental and ancestry informative markers.

    PubMed

    Hollard, Clémence; Keyser, Christine; Giscard, Pierre-Henri; Tsagaan, Turbat; Bayarkhuu, Noost; Bemmann, Jan; Crubézy, Eric; Ludes, Bertrand

    2014-09-01

    The Altai Mountains have been a long-term boundary zone between the Eurasian Steppe populations and South and East Asian populations. To disentangle some of the historical population movements in this area, 14 ancient human specimens excavated in the westernmost part of the Mongolian Altai were studied. Thirteen of them were dated from the Middle to the End of the Bronze Age and one of them to the Eneolithic period. The environmental conditions encountered in this region led to the good preservation of DNA in the human remains. Therefore, a multi-markers approach was adopted for the genetic analysis of identity, ancestry and phenotype markers. Mitochondrial DNA analyses revealed that the ancient Altaians studied carried both Western (H, U, T) and Eastern (A, C, D) Eurasian lineages. In the same way, the patrilineal gene pool revealed the presence of different haplogroups (Q1a2a1-L54, R1a1a1b2-Z93 and C), probably marking different origins for the male paternal lineages. To go further in the search of the origin of these ancient specimens, phenotypical characters (i.e. hair and eye color) were determined. For this purpose, we adapted the HIrisPlex assay recently described to MALDI-TOF mass spectrometry. In addition, some ancestry informative markers were analyzed with this assay. The results revealed mixed phenotypes among this group confirming the probable admixed ancestry of the studied Altaian population at the Middle Bronze Age. The good results obtained from ancient DNA samples suggest that this approach might be relevant for forensic casework too.

  11. Photochemical mutagenesis: examples and toxicological relevance.

    PubMed

    Gocke, E

    2001-01-01

    Induction of DNA damage as a consequence of exposure to UV light has been established as the major cause of skin cancer. DNA molecules absorb photon energy directly for wavelengths <320 nm, and lead to well-characterized mutagenic DNA damage. Alternatively, endogenous or exogenous chemicals (sensitizers) may absorb light with the potential of subsequent energy or electron transfer, and lead indirectly to DNA damage. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably, psoralen and chlorpromazine derivatives have been established as photomutagens and the reaction mechanisms have been identified; the fluoroquinolone antibiotics have more recently been recognized as being photomutagenic. The type of DNA damage and the modulation by antioxidants indicate the involvement of reactive oxygen species (ROS), but other mechanisms are also reported for, at least, some derivatives. In routine genotoxicity studies, we observed the photomutagenic activity of a compound (Ro 19-8022) under development as an anxiolytic agent in the Ames tester strain TA102 under normal laboratory illumination conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The finding led to the termination of drug development. Another example of a pharmaceutical for which photogenotoxic properties were observed during development is Ro 47-7737, a bisquinoline derivative of the antimalaria compound chloroquine. Also in this case, the photochemical reactivity contributed to the termination of the development process. The risk/benefit assessment for the described compounds has to take into account the human exposure situation, for example, the ability to avoid light exposure during treatment. Consideration of photochemical mutagenesis is specifically important for sunscreen ingredients. The active components of sunscreen lotions are efficient UV absorbers. Consequently

  12. Use of Mutagenesis, Genetic Mapping and Next Generation Transcriptomics to Investigate Insecticide Resistance Mechanisms

    PubMed Central

    Kalajdzic, Predrag; Oehler, Stefan; Reczko, Martin; Pavlidi, Nena; Vontas, John; Hatzigeorgiou, Artemis G.; Savakis, Charalambos

    2012-01-01

    Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w−]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results. PMID:22768270

  13. Predicting oligonucleotide-directed mutagenesis failures in protein engineering

    PubMed Central

    Wassman, Christopher D.; Tam, Phillip Y.; Lathrop, Richard H.; Weiss, Gregory A.

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed ‘cross-hybridization’, as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries. PMID:15585664

  14. Integrated Analysis of Transcriptomic and Proteomic Datasets Reveals Information on Protein Expressivity and Factors Affecting Translational Efficiency.

    PubMed

    Wang, Jiangxin; Wu, Gang; Chen, Lei; Zhang, Weiwen

    2016-01-01

    Integrated analysis of large-scale transcriptomic and proteomic data can provide important insights into the metabolic mechanisms underlying complex biological systems. In this chapter, we present methods to address two aspects of issues related to integrated transcriptomic and proteomic analysis. First, due to the fact that proteomic datasets are often incomplete, and integrated analysis of partial proteomic data may introduce significant bias. To address these issues, we describe a zero-inflated Poisson (ZIP)-based model to uncover the complicated relationships between protein abundances and mRNA expression levels, and then apply them to predict protein abundance for the proteins not experimentally detected. The ZIP model takes into consideration the undetected proteins by assuming that there is a probability mass at zero representing expressed proteins that were undetected owing to technical limitations. The model validity is demonstrated using biological information of operons, regulons, and pathways. Second, weak correlation between transcriptomic and proteomic datasets is often due to biological factors affecting translational processes. To quantify the effects of these factors, we describe a multiple regression-based statistical framework to quantitatively examine the effects of various translational efficiency-related sequence features on mRNA-protein correlation. Using the datasets from sulfate-reducing bacteria Desulfovibrio vulgaris, the analysis shows that translation-related sequence features can contribute up to 15.2-26.2% of the total variation of the correlation between transcriptomic and proteomic datasets, and also reveals the relative importance of various features in translation process.

  15. Stabilization of a prokaryotic LAT transporter by random mutagenesis

    PubMed Central

    Rodríguez-Banqueri, Arturo; Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Kowalczyk, Lukasz; Perálvarez-Marín, Alex

    2016-01-01

    The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest. PMID:26976827

  16. Genetic aspects of targeted insertion mutagenesis in yeasts.

    PubMed

    Klinner, U; Schäfer, B

    2004-05-01

    Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

  17. Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics1[W][OA

    PubMed Central

    Cui, Yaya; Barampuram, Shyam; Stacey, Minviluz G.; Hancock, C. Nathan; Findley, Seth; Mathieu, Melanie; Zhang, Zhanyuan; Parrott, Wayne A.; Stacey, Gary

    2013-01-01

    Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean. PMID:23124322

  18. ENU mutagenesis to generate genetically modified rat models.

    PubMed

    van Boxtel, Ruben; Gould, Michael N; Cuppen, Edwin; Smits, Bart M G

    2010-01-01

    The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism.

  19. Self vs. other: neural correlates underlying agent identification based on unimodal auditory information as revealed by electrotomography (sLORETA).

    PubMed

    Justen, C; Herbert, C; Werner, K; Raab, M

    2014-02-14

    Recent neuroscientific studies have identified activity changes in an extensive cerebral network consisting of medial prefrontal cortex, precuneus, temporo-parietal junction, and temporal pole during the perception and identification of self- and other-generated stimuli. Because this network is supposed to be engaged in tasks which require agent identification, it has been labeled the evaluation network (e-network). The present study used self- versus other-generated movement sounds (long jumps) and electroencephalography (EEG) in order to unravel the neural dynamics of agent identification for complex auditory information. Participants (N=14) performed an auditory self-other identification task with EEG. Data was then subjected to a subsequent standardized low-resolution brain electromagnetic tomography (sLORETA) analysis (source localization analysis). Differences between conditions were assessed using t-statistics (corrected for multiple testing) on the normalized and log-transformed current density values of the sLORETA images. Three-dimensional sLORETA source localization analysis revealed cortical activations in brain regions mostly associated with the e-network, especially in the medial prefrontal cortex (bilaterally in the alpha-1-band and right-lateralized in the gamma-band) and the temporo-parietal junction (right hemisphere in the alpha-1-band). Taken together, the findings are partly consistent with previous functional neuroimaging studies investigating unimodal visual or multimodal agent identification tasks (cf. e-network) and extent them to the auditory domain. Cortical activations in brain regions of the e-network seem to have functional relevance, especially the significantly higher cortical activation in the right medial prefrontal cortex.

  20. Coupled mutagenesis screens and genetic mapping in zebrafish.

    PubMed Central

    Rawls, John F; Frieda, Matthew R; McAdow, Anthony R; Gross, Jason P; Clayton, Chad M; Heyen, Candy K; Johnson, Stephen L

    2003-01-01

    Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. PMID:12663538

  1. A simple mutagenesis using natural competence in Tannerella forsythia.

    PubMed

    Nishikawa, Kiyoshi; Tanaka, Yoshinobu

    2013-09-01

    We report the discovery of natural competence in Tannerella forsythia and its application to targeted chromosomal mutagenesis. Keeping T. forsythia in a biofilm throughout the procedure allowed efficient DNA uptake and allelic replacement. This simple method is cost-effective and reproducible compared with the conventional protocols using broth culture and electroporation.

  2. Favipiravir elicits antiviral mutagenesis during virus replication in vivo

    PubMed Central

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-01-01

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses. DOI: http://dx.doi.org/10.7554/eLife.03679.001 PMID:25333492

  3. What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

    ERIC Educational Resources Information Center

    Linde, Ana R.; Garcia-Vazquez, Eva

    2009-01-01

    The micronucleus test is widely employed in environmental health research. It can also be an excellent tool for learning important concepts in environmental health. In this article we present an inquiry-based laboratory exercise where students explore several theoretical and practical aspects of environmental mutagenesis employing the micronucleus…

  4. Methods for targetted mutagenesis in gram-positive bacteria

    DOEpatents

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  5. Functional MRI Representational Similarity Analysis Reveals a Dissociation between Discriminative and Relative Location Information in the Human Visual System.

    PubMed

    Roth, Zvi N

    2016-01-01

    Neural responses in visual cortex are governed by a topographic mapping from retinal locations to cortical responses. Moreover, at the voxel population level early visual cortex (EVC) activity enables accurate decoding of stimuli locations. However, in many cases information enabling one to discriminate between locations (i.e., discriminative information) may be less relevant than information regarding the relative location of two objects (i.e., relative information). For example, when planning to grab a cup, determining whether the cup is located at the same retinal location as the hand is hardly relevant, whereas the location of the cup relative to the hand is crucial for performing the action. We have previously used multivariate pattern analysis techniques to measure discriminative location information, and found the highest levels in EVC, in line with other studies. Here we show, using representational similarity analysis, that availability of discriminative information in fMRI activation patterns does not entail availability of relative information. Specifically, we find that relative location information can be reliably extracted from activity patterns in posterior intraparietal sulcus (pIPS), but not from EVC, where we find the spatial representation to be warped. We further show that this variability in relative information levels between regions can be explained by a computational model based on an array of receptive fields. Moreover, when the model's receptive fields are extended to include inhibitory surround regions, the model can account for the spatial warping in EVC. These results demonstrate how size and shape properties of receptive fields in human visual cortex contribute to the transformation of discriminative spatial representations into relative spatial representations along the visual stream.

  6. Functional MRI Representational Similarity Analysis Reveals a Dissociation between Discriminative and Relative Location Information in the Human Visual System

    PubMed Central

    Roth, Zvi N.

    2016-01-01

    Neural responses in visual cortex are governed by a topographic mapping from retinal locations to cortical responses. Moreover, at the voxel population level early visual cortex (EVC) activity enables accurate decoding of stimuli locations. However, in many cases information enabling one to discriminate between locations (i.e., discriminative information) may be less relevant than information regarding the relative location of two objects (i.e., relative information). For example, when planning to grab a cup, determining whether the cup is located at the same retinal location as the hand is hardly relevant, whereas the location of the cup relative to the hand is crucial for performing the action. We have previously used multivariate pattern analysis techniques to measure discriminative location information, and found the highest levels in EVC, in line with other studies. Here we show, using representational similarity analysis, that availability of discriminative information in fMRI activation patterns does not entail availability of relative information. Specifically, we find that relative location information can be reliably extracted from activity patterns in posterior intraparietal sulcus (pIPS), but not from EVC, where we find the spatial representation to be warped. We further show that this variability in relative information levels between regions can be explained by a computational model based on an array of receptive fields. Moreover, when the model's receptive fields are extended to include inhibitory surround regions, the model can account for the spatial warping in EVC. These results demonstrate how size and shape properties of receptive fields in human visual cortex contribute to the transformation of discriminative spatial representations into relative spatial representations along the visual stream. PMID:27242455

  7. Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

    PubMed

    Denzin, L K; Voss, E W

    1992-05-05

    In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active

  8. Reconstitutional Mutagenesis of the Maize P Gene by Short-Range Ac Transpositions

    PubMed Central

    Moreno, M. A.; Chen, J.; Greenblatt, I.; Dellaporta, S. L.

    1992-01-01

    The tendency for Ac to transpose over short intervals has been utilized to develop insertional mutagenesis and fine structure genetic mapping strategies in maize. We recovered excisions of Ac from the P gene and insertions into nearby chromosomal sites. These closely linked Ac elements reinserted into the P gene, reconstituting over 250 unstable variegated alleles. Reconstituted alleles condition a variety of variegation patterns that reflect the position and orientation of Ac within the P gene. Molecular mapping and DNA sequence analyses have shown that reinsertion sites are dispersed throughout a 12.3-kb chromosomal region in the promoter, exons and introns of the P gene, but in some regions insertions sites were clustered in a nonrandom fashion. Transposition profiles and target site sequence data obtained from these studies have revealed several features of Ac transposition including its preference for certain target sites. These results clearly demonstrate the tendency of Ac to transpose to nearby sites in both proximal and distal directions from the donor site. With minor modifications, reconstitutional mutagenesis should be applicable to many Ac-induced mutations in maize and in other plant species and can possibly be extended to other eukaryotic transposon systems as well. PMID:1325389

  9. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling.

    PubMed

    Hackel, Benjamin J; Kapila, Atul; Wittrup, K Dane

    2008-09-19

    The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino acid composition and loop length. The library was screened by fluorescence-activated cell sorting to isolate binders to lysozyme. An affinity maturation scheme was developed to rapidly and broadly diversify populations of clones by random mutagenesis as well as homologous recombination-driven shuffling of mutagenized loops. The novel library and affinity maturation scheme combined to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A secondary affinity maturation identified a stable 1.1 pM binder, the highest affinity yet reported for an Fn3 domain. In addition to extension of the affinity limit for this scaffold, the results demonstrate the ability to achieve high-affinity binding while preserving stability and the monomeric state. This library design and affinity maturation scheme is highly efficient, utilizing an initial diversity of 2x10(7) clones and screening only 1x10(8) mutants (totaled over all affinity maturation libraries). Analysis of intermediate populations revealed that loop length diversity, loop shuffling, and recursive mutagenesis of diverse populations are all critical components.

  10. Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

    SciTech Connect

    Kermany, Mohammad; Parker, Lisan; Guo, Yun-Kai; Miller, Darla R; Swanson, Douglas J; Yoo, Tai-June; Goldowitz, Daniel; Zuo, Jian

    2006-01-01

    The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

  11. Mutagenesis Is Elevated in Male Germ Cells Obtained from DNA Polymerase-beta Heterozygous Mice1

    PubMed Central

    Allen, Diwi; Herbert, Damon C.; McMahan, C. Alex; Rotrekl, Vladimir; Sobol, Robert W.; Wilson, Samuel H.; Walter, Christi A.

    2008-01-01

    Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency.. PMID:18650495

  12. Rank Order Coding: a Retinal Information Decoding Strategy Revealed by Large-Scale Multielectrode Array Retinal Recordings123

    PubMed Central

    Maccione, Alessandro; Di Marco, Stefano; Kornprobst, Pierre

    2016-01-01

    How a population of retinal ganglion cells (RGCs) encodes the visual scene remains an open question. Going beyond individual RGC coding strategies, results in salamander suggest that the relative latencies of a RGC pair encode spatial information. Thus, a population code based on this concerted spiking could be a powerful mechanism to transmit visual information rapidly and efficiently. Here, we tested this hypothesis in mouse by recording simultaneous light-evoked responses from hundreds of RGCs, at pan-retinal level, using a new generation of large-scale, high-density multielectrode array consisting of 4096 electrodes. Interestingly, we did not find any RGCs exhibiting a clear latency tuning to the stimuli, suggesting that in mouse, individual RGC pairs may not provide sufficient information. We show that a significant amount of information is encoded synergistically in the concerted spiking of large RGC populations. Thus, the RGC population response described with relative activities, or ranks, provides more relevant information than classical independent spike count- or latency- based codes. In particular, we report for the first time that when considering the relative activities across the whole population, the wave of first stimulus-evoked spikes is an accurate indicator of stimulus content. We show that this coding strategy coexists with classical neural codes, and that it is more efficient and faster. Overall, these novel observations suggest that already at the level of the retina, concerted spiking provides a reliable and fast strategy to rapidly transmit new visual scenes. PMID:27275008

  13. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    PubMed Central

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  14. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

    SciTech Connect

    Wang, Weina; Hellinga, Homme W.; Beese, Lorena S.

    2012-05-10

    Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C {center_dot} A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

  15. Targeted mutagenesis in sea urchin embryos using TALENs.

    PubMed

    Hosoi, Sayaka; Sakuma, Tetsushi; Sakamoto, Naoaki; Yamamoto, Takashi

    2014-01-01

    Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos.

  16. Insertional mutagenesis of an industrial strain of Streptococcus thermophilus.

    PubMed

    Labarre, C; Schirawski, J; van der Zwet, A; Fitzgerald, G F; van Sinderen, D

    2001-06-12

    Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system. The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep(-)) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Rep(ts)) helper plasmid pVE6007. In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism. Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation.

  17. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  18. Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

    PubMed Central

    Tascon, R I; Rodriguez-Ferri, E F; Gutierrez-Martin, C B; Rodriguez-Barbosa, I; Berche, P; Vazquez-Boland, J A

    1993-01-01

    A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed. Images PMID:8396122

  19. Use of liver cell cultures in mutagenesis studies

    SciTech Connect

    Huberman, E.; Jones, C.A.

    1980-09-30

    A sensitive cell-mediated assay has been developed for testing the mutagenesis of liver carcinogens. Mutagenesis was detected in Chinese hamster V79 cells that were cocultivated with hepatocytes isolated after collagenase/hyaluronidase digestion of rat liver slices. Mutations were characterized by resistance to ouabain and 6-thioguanine. Seven of the nitrosamines, which are potent liver carcinogens, exhibited a mutagenic response. Mutagenesis with these carcinogens could be detected at ..mu..molar doses. The polyaromatic hydrocarbon benzo(a)pyrene, which is not a liver carcinogen, but can cause fibrosarcomas, was not mutagenic in this assay, but was mutagenic in a fibroblast-mediated assay. The liver carcinogen, aflatoxin B/sub 1/, which usually does not induce fibrosarcomas, exhibited an inverse situation; it was mutagenic for V79 cells in the presence of liver cells but not in the presence of fibroblasts. We suggest that the use of various cell types, including hepatocytes prepared by the slicing method for carcinogen metabolism, and mutable V79 cells offers a sensitive assay for determining the mutagenic potential of chemical carcinogens, and may also allow a study of their organ specificity.

  20. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    SciTech Connect

    Puck, T.P.; Johnson, R.; Waldren, C.A. ); Morse, H. )

    1993-09-01

    The well-known action of caffeine in synergizing mutagenesis (including chromosome aberrations) of agents like ionizing radiation by inhibition of cellular repair processes has been incorporated into a rapid procedure for detection of mutagenicity with high sensitivity. Effects of 5-10 rads of [gamma]-irradiation, which approximate the human lifetime dose accumulation from background radiation, can be detected in a two-day procedure using an immortalized human WBC culture. Chromosomally visible lesions are scored on cells incubated for 2 h after irradiation in the presence and absence of 1.0 mg/ml of caffeine. An eightfold amplification of scorable lesions is achieved over the action of radiation alone. This approach provides a closer approximation to absolute mutagenicity unmitigated by repair processes, which can vary in different situations. It is proposed that mutagenesis testing of this kind, using caffiene or other repair-inhibitory agents, be employed to identify mutagens in their effective concentrations to which human populations may be exposed; to detect agents such as caffeine that may synergize mutagenic actions and pose epidemiologic threats; and to discover effective anti-mutagens. Information derived from the use of such procedures may help prevent cancer and newly acquired genetic disease.

  1. Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

    PubMed

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

    2016-01-01

    A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

  2. Brain-based decoding of mentally imagined film clips and sounds reveals experience-based information patterns in film professionals.

    PubMed

    de Borst, Aline W; Valente, Giancarlo; Jääskeläinen, Iiro P; Tikka, Pia

    2016-04-01

    In the perceptual domain, it has been shown that the human brain is strongly shaped through experience, leading to expertise in highly-skilled professionals. What has remained unclear is whether specialization also shapes brain networks underlying mental imagery. In our fMRI study, we aimed to uncover modality-specific mental imagery specialization of film experts. Using multi-voxel pattern analysis we decoded from brain activity of professional cinematographers and sound designers whether they were imagining sounds or images of particular film clips. In each expert group distinct multi-voxel patterns, specific for the modality of their expertise, were found during classification of imagery modality. These patterns were mainly localized in the occipito-temporal and parietal cortex for cinematographers and in the auditory cortex for sound designers. We also found generalized patterns across perception and imagery that were distinct for the two expert groups: they involved frontal cortex for the cinematographers and temporal cortex for the sound designers. Notably, the mental representations of film clips and sounds of cinematographers contained information that went beyond modality-specificity. We were able to successfully decode the implicit presence of film genre from brain activity during mental imagery in cinematographers. The results extend existing neuroimaging literature on expertise into the domain of mental imagery and show that experience in visual versus auditory imagery can alter the representation of information in modality-specific association cortices.

  3. Comparative mutagenesis of human cells in vitro and in vivo. Progress report and financial statement

    SciTech Connect

    Thilly, W.G.

    1983-01-01

    Our laboratory has undertaken the study of mutagenesis from two perspectives. First, we are devising methods to measure mutation in human beings and to recognize specific patterns of mutation indicative of exposures to particular environmental agents. Second, we are studying the basic question of how chemicals induce mutation and also their potential effects on gene regulation and expression. Specifically we report results which show the applicability of measuring mutation frequencies at small loci in a human lymphoblast cell line in the generation of mutational spectra. Our experiments in the area of gene regulation have revealed that 5-azacytidine can reverse the previously discovered effect of BUdR in inducing pseudomutation at the TK locus. In addition, we have developed an improvement in the performance of human lymphoblast mutation assays by devising means to measure plating efficiency at high cell densities.

  4. Germ cell comparative Drosophila mutagenesis: sensitivity and mutation pattern in chemically treated stem cells

    SciTech Connect

    Abrahamson, S.; Houtchens, K.; Li Jia, X.; Foureman, P.

    1983-01-01

    Mutagenesis studies on Drosophila oogonial cells with methylnitrosourea, dimethylnitrosamine, and diethylnitrosamine revealed unexpectedly high rates of sex-linked recessive lethals relative to other male and female germ cell stages. Indeed, the oogonial mutation rates with chemicals are higher than with massive x-ray or neutron exposures of oogonia. Analysis of the distribution of lethals per treated female suggests most of the mutations recovered are of independent origin, with very small levels of clustering of identical mutations. In the male stem cell population (spermatogonia) on the other hand, the distribution of lethals is primarily nonrandom and highly clustered. The nature of the mutational endpoint and the different pattern of germ cell development in the two sexes are the probable causes of this difference. The oogonial sensitivity to chemical mutagens may have important bearing on strategies for assessing human hazard.

  5. The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis

    PubMed Central

    Devedjiev, Yancho D.

    2015-01-01

    Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d < 1.5 Å) reveals that in contrast to current views, static disorder and high side-chain entropy are common in the crystal contact area. These observations challenge the validity of the theory that presumes that the occurrence of well ordered patches of side chains at the surface is an essential prerequisite for a successful crystallization event. The present paper provides evidence in support of the approach for understanding protein crystallization as a process dependent on multiple factors, each with its relative contribution, rather than a phenomenon driven by a few dominant physicochemical characteristics. The role of the molecular shape as a factor in the crystallization of proteins by surface mutagenesis is discussed. PMID:25664789

  6. Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach.

    PubMed

    Uytterhoeven, Birgit; Appermans, Kenny; Song, Lijiang; Masschelein, Joleen; Lathouwers, Thomas; Michiels, Chris W; Lavigne, Rob

    2016-04-01

    Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo.

  7. Probabilistic information transmission in a network of coupled oscillators reveals speed-accuracy trade-off in responding to threats

    NASA Astrophysics Data System (ADS)

    Chicoli, Amanda; Paley, Derek A.

    2016-11-01

    Individuals in a group may obtain information from other group members about the environment, including the location of a food source or the presence of a predator. Here, we model how information spreads in a group using a susceptible-infected-removed epidemic model. We apply this model to a simulated shoal of fish using the motion dynamics of a coupled oscillator model, in order to test the biological hypothesis that polarized or aligned shoaling leads to faster and more accurate escape responses. The contributions of this study are the (i) application of a probabilistic model of epidemics to the study of collective animal behavior; (ii) testing the biological hypothesis that group cohesion improves predator escape; (iii) quantification of the effect of social cues on startle propagation; and (iv) investigation of the variation in response based on network connectivity. We find that when perfectly aligned individuals in a group are startled, there is a rapid escape by individuals that directly detect the threat, as well as by individuals responding to their neighbors. However, individuals that are not startled do not head away from the threat. In startled groups that are randomly oriented, there is a rapid, accurate response by individuals that directly detect the threat, followed by less accurate responses by individuals responding to neighbor cues. Over the simulation duration, however, even unstartled individuals head away from the threat. This study illustrates a potential speed-accuracy trade-off in the startle response of animal groups, in agreement with several previous experimental studies. Additionally, the model can be applied to a variety of group decision-making processes, including those involving higher-dimensional motion.

  8. Morphology informed by phylogeny reveals unexpected patterns of species differentiation in the aquatic moss Rhynchostegium riparioides s.l.

    PubMed

    Hutsemékers, Virginie; Vieira, Cristiana C; Ros, Rosa María; Huttunen, Sanna; Vanderpoorten, Alain

    2012-02-01

    Bryophyte floras typically exhibit extremely low levels of endemism. The interpretation, that this might reflect taxonomic shortcomings, is tested here for the Macaronesian flora, using the moss species complex of Rhynchostegium riparioides as a model. The deep polyphyly of R. riparioides across its distribution range reveals active differentiation that better corresponds to geographic than morphological differences. Morphometric analyses are, in fact, blurred by a size gradient that accounts for 80% of the variation observed among gametophytic traits. The lack of endemic diversification observed in R. riparioides in Macaronesia weakens the idea that the low rates of endemism observed in the Macaronesian bryophyte flora might solely be explained by taxonomic shortcomings. To the reverse, the striking polyphyly of North American and European lineages of R. riparioides suggests that the similarity between the floras of these continents has been over-emphasized. Discriminant analyses point to the existence of morphological discontinuities among the lineages resolved by the molecular phylogeny. The global rate of error associated to species identification based on morphology (0.23) indicates, however, that intergradation of shape and size characters among species in the group challenges their identification.

  9. Masked priming of conceptual features reveals differential brain activation during unconscious access to conceptual action and sound information.

    PubMed

    Trumpp, Natalie M; Traub, Felix; Kiefer, Markus

    2013-01-01

    Previous neuroimaging studies suggested an involvement of sensory-motor brain systems during conceptual processing in support of grounded cognition theories of conceptual memory. However, in these studies with visible stimuli, contributions of strategic imagery or semantic elaboration processes to observed sensory-motor activity cannot be entirely excluded. In the present study, we therefore investigated the electrophysiological correlates of unconscious feature-specific priming of action- and sound-related concepts within a novel feature-priming paradigm to specifically probe automatic processing of conceptual features without the contribution of possibly confounding factors such as orthographic similarity or response congruency. Participants were presented with a masked subliminal prime word and a subsequent visible target word. In the feature-priming conditions primes as well as targets belonged to the same conceptual feature dimension (action or sound, e.g., typewriter or radio) whereas in the two non-priming conditions, either the primes or the targets consisted of matched control words with low feature relevance (e.g., butterfly or candle). Event-related potential analyses revealed unconscious feature-specific priming effects at fronto-central electrodes within 100 to 180 ms after target stimulus onset that differed with regard to topography and underlying neural generators. In congruency with previous findings under visible stimulation conditions, these differential subliminal ERP feature-priming effects demonstrate an unconscious automatic access to action versus sound features of concepts. The present results therefore support grounded cognition theory suggesting that activity in sensory and motor areas during conceptual processing can also occur unconsciously and is not mandatorily accompanied by a vivid conscious experience of the conceptual content such as in imagery.

  10. Sandcastle: software for revealing latent information in multiple experimental ChIP-chip datasets via a novel normalisation procedure

    PubMed Central

    Bennett, Mark; Evans, Katie Ellen; Yu, Shirong; Teng, Yumin; Webster, Richard M.; Powell, James; Waters, Raymond; Reed, Simon H.

    2015-01-01

    ChIP-chip is a microarray based technology for determining the genomic locations of chromatin bound factors of interest, such as proteins. Standard ChIP-chip analyses employ peak detection methodologies to generate lists of genomic binding sites. No previously published method exists to enable comparative analyses of enrichment levels derived from datasets examining different experimental conditions. This restricts the use of the technology to binary comparisons of presence or absence of features between datasets. Here we present the R package Sandcastle — Software for the Analysis and Normalisation of Data from ChIP-chip AssayS of Two or more Linked Experiments — which allows for comparative analyses of data from multiple experiments by normalising all datasets to a common background. Relative changes in binding levels between experimental datasets can thus be determined, enabling the extraction of latent information from ChIP-chip experiments. Novel enrichment detection and peak calling algorithms are also presented, with a range of graphical tools, which facilitate these analyses. The software and documentation are available for download from http://reedlab.cardiff.ac.uk/sandcastle. PMID:26307543

  11. Pyrosequencing: Applicability for Studying DNA Damage-induced Mutagenesis

    PubMed Central

    Minko, Irina G.; Earley, Lauriel F.; Larlee, Kimberly E.; Lin, Ying-Chih; Lloyd, R. Stephen

    2014-01-01

    Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a predetermined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively-labeled probes, and verification of mutations by Sanger sequencing. In search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N6-(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5′-GCNGG-3′ sequence context. Pyrosequencing detected ~8% G to T transversions and ~3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure [Earley et al., 2013]. However, ~3.5% G to C transversions and ~2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ~1-2% for A and T and ~5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols. PMID:24962778

  12. EMS Mutagenesis in the Pea Aphid Acyrthosiphon pisum

    PubMed Central

    Tagu, Denis; Le Trionnaire, Gaël; Tanguy, Sylvie; Gauthier, Jean-Pierre; Huynh, Jean-René

    2014-01-01

    In aphids, clonal individuals can show distinct morphologic traits in response to environmental cues. Such phenotypic plasticity cannot be studied with classical genetic model organisms such as Caenorhabditis elegans or Drosophila melanogaster. The genetic basis of this biological process remain unknown, as mutations affecting this process are not available in aphids. Here, we describe a protocol to treat third-stage larvae with an alkylating mutagen, ethyl methanesulfonate (EMS), to generate random mutations within the Acyrthosiphon pisum genome. We found that even low concentrations of EMS were toxic for two genotypes of A. pisum. Mutagenesis efficiency was nevertheless assessed by estimating the occurrence of mutational events on the X chromosome. Indeed, any lethal mutation on the X-chromosome would kill males that are haploid on the X so that we used the proportion of males as an estimation of mutagenesis efficacy. We could assess a putative mutation rate of 0.4 per X-chromosome at 10 mM of EMS. We then applied this protocol to perform a small-scale mutagenesis on parthenogenetic individuals, which were screened for defects in their ability to produce sexual individuals in response to photoperiod shortening. We found one mutant line showing a reproducible altered photoperiodic response with a reduced production of males and the appearance of aberrant winged males (wing atrophy, alteration of legs morphology). This mutation appeared to be stable because it could be transmitted over several generations of parthenogenetic individuals. To our knowledge, this study represents the first example of an EMS-generated aphid mutant. PMID:24531730

  13. Pollen tetrads in the detection of environmental mutagenesis

    SciTech Connect

    Mulcahy, D.L.

    1981-01-01

    Although pollen is a very sensitive indicator of environmental mutagenesis, it is also sensitive to nonmutagenic environmental stress. By analyzing pollen tetrads, rather than individual pollen grains, it is possible to distinguish between mutagenic and nonmutagenic influences. Another advantage of using pollen tetrads in mutagenicity studies is that it is possible to discriminate between pre- and post-pachytene mutations. This eliminates the mutant sector problem of a single mutational event giving rise to a large number of mutant cells. Methods of analyzing pollen tetrads are described.

  14. Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

    PubMed

    Hayashi, Toshinori; Takeuchi, Takashi

    2016-01-01

    Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts.

  15. Chemical mutagenesis: an emerging issue for public health.

    PubMed Central

    Claxton, L D; Barry, P Z

    1977-01-01

    Chemical mutagens are recognized as prevalent in the environment and a potential threat to the health of future generations. This paper presents an overview of chemical mutagenesis as an issue for public health. Several problems in the determination of risk to human populations are discussed, including difficulties of extrapolating scientific data to humans, the latency period between exposure and recognizable genetic damage, and the large number of chemicals which must be tested. Test systems are described. Possibilities of control through federal regulation are discussed. PMID:911015

  16. Spectrum of Bmp5 mutations from germline mutagenesis experiments in mice

    SciTech Connect

    Marker, P.C.; Kwonjune Seung; Bland, A.E.

    1997-02-01

    Over 40 years of mutagenesis experiments using the mouse specific-locus test have produced a large number of induced germline mutations at seven loci, among them the short ear locus. We have previously shown that the short ear locus encodes bone morphogenetic protein 5 (BMP5), a member of a large family of secreted signaling molecules that play key roles in axis formation, tissue differentiation, mesenchymal-epithelial interactions, and skeletal development. Here we examine 24 chemical- and radiation-induced mutations at the short ear locus. Sequence changes in the Bmp5 open reading frame confirm the importance of cysteine residues in the function of TGF{beta} superfamily members. The spectrum of N-ethyl-N-nitrosourea-induced mutations also provides new information about the basepair, sequence context, and strand specificity of germline mutations in mammals. 52 refs., 3 figs., 2 tabs.

  17. Spectrum of Bmp5 Mutations from Germline Mutagenesis Experiments in Mice

    PubMed Central

    Marker, P. C.; Seung, K.; Bland, A. E.; Russell, L. B.; Kingsley, D. M.

    1997-01-01

    Over 40 years of mutagenesis experiments using the mouse specific-locus test have produced a large number of induced germline mutations at seven loci, among them the short ear locus. We have previously shown that the short ear locus encodes bone morphogenetic protein 5 (BMP5), a member of a large family of secreted signaling molecules that play key roles in axis formation, tissue differentiation, mesenchymal-epithelial interactions, and skeletal development. Here we examine 24 chemical- and radiation-induced mutations at the short ear locus. Sequence changes in the Bmp5 open reading frame confirm the importance of cysteine residues in the function of TGFβ superfamily members. The spectrum of N-ethyl-N-nitrosourea-induced mutations also provides new information about the basepair, sequence context, and strand specificity of germline mutations in mammals. PMID:9071596

  18. Causal relationships between frequency bands of extracellular signals in visual cortex revealed by an information theoretic analysis.

    PubMed

    Besserve, Michel; Schölkopf, Bernhard; Logothetis, Nikos K; Panzeri, Stefano

    2010-12-01

    Characterizing how different cortical rhythms interact and how their interaction changes with sensory stimulation is important to gather insights into how these rhythms are generated and what sensory function they may play. Concepts from information theory, such as Transfer Entropy (TE), offer principled ways to quantify the amount of causation between different frequency bands of the signal recorded from extracellular electrodes; yet these techniques are hard to apply to real data. To address the above issues, in this study we develop a method to compute fast and reliably the amount of TE from experimental time series of extracellular potentials. The method consisted in adapting efficiently the calculation of TE to analog signals and in providing appropriate sampling bias corrections. We then used this method to quantify the strength and significance of causal interaction between frequency bands of field potentials and spikes recorded from primary visual cortex of anaesthetized macaques, both during spontaneous activity and during binocular presentation of naturalistic color movies. Causal interactions between different frequency bands were prominent when considering the signals at a fine (ms) temporal resolution, and happened with a very short (ms-scale) delay. The interactions were much less prominent and significant at coarser temporal resolutions. At high temporal resolution, we found strong bidirectional causal interactions between gamma-band (40-100 Hz) and slower field potentials when considering signals recorded within a distance of 2 mm. The interactions involving gamma bands signals were stronger during movie presentation than in absence of stimuli, suggesting a strong role of the gamma cycle in processing naturalistic stimuli. Moreover, the phase of gamma oscillations was playing a stronger role than their amplitude in increasing causations with slower field potentials and spikes during stimulation. The dominant direction of causality was mainly found

  19. Risk map for cutaneous leishmaniasis in Ethiopia based on environmental factors as revealed by geographical information systems and statistics.

    PubMed

    Seid, Ahmed; Gadisa, Endalamaw; Tsegaw, Teshome; Abera, Adugna; Teshome, Aklilu; Mulugeta, Abate; Herrero, Merce; Argaw, Daniel; Jorge, Alvar; Kebede, Asnakew; Aseffa, Abraham

    2014-05-01

    Cutaneous leishmaniasis (CL) is a neglected tropical disease strongly associated with poverty. Treatment is problematic and no vaccine is available. Ethiopia has seen new outbreaks in areas previously not known to be endemic, often with co-infection by the human immunodeficiency virus (HIV) with rates reaching 5.6% of the cases. The present study concerns the development of a risk model based on environmental factors using geographical information systems (GIS), statistical analysis and modelling. Odds ratio (OR) of bivariate and multivariate logistic regression was used to evaluate the relative importance of environmental factors, accepting P ≤ 0.056 as the inclusion level for the model's environmental variables. When estimating risk from the viewpoint of geographical surface, slope, elevation and annual rainfall were found to be good predictors of CL presence based on both probabilistic and weighted overlay approaches. However, when considering Ethiopia as whole, a minor difference was observed between the two methods with the probabilistic technique giving a 22.5% estimate, while that of weighted overlay approach was 19.5%. Calculating the population according to the land surface estimated by the latter method, the total Ethiopian population at risk for CL was estimated at 28,955,035, mainly including people in the highlands of the regional states of Amhara, Oromia, Tigray and the Southern Nations, Nationalities and Peoples' Region, one of the nine ethnic divisions in Ethiopia. Our environmental risk model provided an overall prediction accuracy of 90.4%. The approach proposed here can be replicated for other diseases to facilitate implementation of evidence-based, integrated disease control activities.

  20. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

    PubMed Central

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  1. Mutagenesis of conserved amino acids of Helicobacter pylori fur reveals residues important for function.

    PubMed

    Carpenter, Beth M; Gancz, Hanan; Benoit, Stéphane L; Evans, Sarah; Olsen, Cara H; Michel, Sarah L J; Maier, Robert J; Merrell, D Scott

    2010-10-01

    The ferric uptake regulator (Fur) of the medically important pathogen Helicobacter pylori is unique in that it has been shown to function as a repressor both in the presence of an Fe2+ cofactor and in its apo (non-Fe2+-bound) form. However, virtually nothing is known concerning the amino acid residues that are important for Fur functioning. Therefore, mutations in six conserved amino acid residues of H. pylori Fur were constructed and analyzed for their impact on both iron-bound and apo repression. In addition, accumulation of the mutant proteins, protein secondary structure, DNA binding ability, iron binding capacity, and the ability to form higher-order structures were also examined for each mutant protein. While none of the mutated residues completely abrogated the function of Fur, we were able to identify residues that were critical for both iron-bound and apo-Fur repression. One mutation, V64A, did not alter regulation of any target genes. However, each of the five remaining mutations showed an effect on either iron-bound or apo regulation. Of these, H96A, E110A, and E117A mutations altered iron-bound Fur regulation and were all shown to influence iron binding to different extents. Additionally, the H96A mutation was shown to alter Fur oligomerization, and the E110A mutation was shown to impact oligomerization and DNA binding. Conversely, the H134A mutant exhibited changes in apo-Fur regulation that were the result of alterations in DNA binding. Although the E90A mutant exhibited alterations in apo-Fur regulation, this mutation did not affect any of the assessed protein functions. This study is the first for H. pylori to analyze the roles of specific amino acid residues of Fur in function and continues to highlight the complexity of Fur regulation in this organism.

  2. Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

    PubMed

    Hingston, Patricia A; Piercey, Marta J; Truelstrup Hansen, Lisbeth

    2015-08-15

    Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.

  3. Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation

    PubMed Central

    Tang, Matthew Y.; Vranas, Marta; Krahn, Andrea I.; Pundlik, Shayal; Trempe, Jean- François; Fon, Edward A.

    2017-01-01

    Parkin and PINK1 function in a common pathway to clear damaged mitochondria. Parkin exists in an auto-inhibited conformation stabilized by multiple interdomain interactions. The binding of PINK1-generated phospho-ubiquitin and the phosphorylation of the ubiquitin-like (Ubl) domain of Parkin at Ser65 release its auto-inhibition, but how and when these events take place in cells remain to be defined. Here we show that mutations that we designed to activate Parkin by releasing the Repressor Element of Parkin (REP) domain, or by disrupting the interface between the RING0:RING2 domains, can completely rescue mutations in the Parkin Ubl that are defective in mitochondrial autophagy. Using a FRET reporter assay we show that Parkin undergoes a conformational change upon phosphorylation that can be mimicked by mutating Trp403 in the REP. We propose a hierarchical model whereby pUb binding on mitochondria enables Parkin phosphorylation, which, in turn, leads to REP removal, E3 ligase activation and mitophagy. PMID:28276439

  4. NbIT - A New Information Theory-Based Analysis of Allosteric Mechanisms Reveals Residues that Underlie Function in the Leucine Transporter LeuT

    PubMed Central

    LeVine, Michael V.; Weinstein, Harel

    2014-01-01

    Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems. PMID:24785005

  5. NbIT--a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT.

    PubMed

    LeVine, Michael V; Weinstein, Harel

    2014-05-01

    Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems.

  6. Heritable site-specific mutagenesis using TALENs in maize.

    PubMed

    Char, Si Nian; Unger-Wallace, Erica; Frame, Bronwyn; Briggs, Sarah A; Main, Marcy; Spalding, Martin H; Vollbrecht, Erik; Wang, Kan; Yang, Bing

    2015-09-01

    Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

  7. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir.

    PubMed

    de Ávila, Ana I; Gallego, Isabel; Soria, Maria Eugenia; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M; Domingo, Esteban; Perales, Celia

    2016-01-01

    Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens.

  8. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir

    PubMed Central

    de Ávila, Ana I.; Gallego, Isabel; Soria, Maria Eugenia; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Domingo, Esteban; Perales, Celia

    2016-01-01

    Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens. PMID:27755573

  9. Mutagenesis of diploid mammalian genes by gene entrapment

    PubMed Central

    Lin, Qing; Donahue, Sarah L.; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B.; Ruley, H. Earl

    2006-01-01

    The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells. PMID:17062627

  10. Mutagenesis of diploid mammalian genes by gene entrapment.

    PubMed

    Lin, Qing; Donahue, Sarah L; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B; Ruley, H Earl

    2006-01-01

    The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector-cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 x 10(-5) to 1.2 x 10(-4) per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells.

  11. TALEN mediated somatic mutagenesis in murine models of cancer

    PubMed Central

    Zhang, Shuyuan; Li, Lin; Kendrick, Sara L.; Gerard, Robert D.; Zhu, Hao

    2014-01-01

    Cancer genome sequencing has identified numerous somatic mutations whose biological relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. TALEN were designed against β-catenin (Ctnnb1) and Apc, two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked if these TALENs could create targeted somatic mutations after hydrodynamic transfection (HDT) into mouse liver. TALENs targeting β-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity (LOH), and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. PMID:25070752

  12. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    PubMed Central

    Bacolla, Albino; Cooper, David N.; Vasquez, Karen M.

    2014-01-01

    Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs). Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS) and other endogenous or exogenous electron-abstracting molecules. PMID:24705290

  13. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  14. Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification

    PubMed Central

    Chen, Liming; Jenjaroenpun, Piroon; Pillai, Andrea Mun Ching; Ivshina, Anna V.; Ow, Ghim Siong; Efthimios, Motakis; Zhiqun, Tang; Lee, Song-Choon; Rogers, Keith; Ward, Jerrold M.; Mori, Seiichi; Adams, David J.; Jenkins, Nancy A.; Copeland, Neal G.; Ban, Kenneth Hon-Kim; Kuznetsov, Vladimir A.; Thiery, Jean Paul

    2017-01-01

    Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers. PMID:28251929

  15. Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification.

    PubMed

    Chen, Liming; Jenjaroenpun, Piroon; Pillai, Andrea Mun Ching; Ivshina, Anna V; Ow, Ghim Siong; Efthimios, Motakis; Zhiqun, Tang; Tan, Tuan Zea; Lee, Song-Choon; Rogers, Keith; Ward, Jerrold M; Mori, Seiichi; Adams, David J; Jenkins, Nancy A; Copeland, Neal G; Ban, Kenneth Hon-Kim; Kuznetsov, Vladimir A; Thiery, Jean Paul

    2017-03-14

    Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers.

  16. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome.

  17. Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

    SciTech Connect

    Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira; Mesecar, Andrew D.

    2010-11-09

    An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 C and withstanding incubation at temperatures up to 70 C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 {angstrom}. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those

  18. Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method

    PubMed Central

    Wang, Yuru; Beal, Peter A.

    2016-01-01

    Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5′ side of the modification site. This 5′ binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure–activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5′ binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure–function relationships in ADAR reactions. PMID:27614075

  19. Site-directed mutagenesis and saturation mutagenesis for the functional study of transcription factors involved in plant secondary metabolite biosynthesis.

    PubMed

    Pattanaik, Sitakanta; Werkman, Joshua R; Kong, Que; Yuan, Ling

    2010-01-01

    Regulation of gene expression is largely coordinated by a complex network of interactions between transcription factors (TFs), co-factors, and their cognate cis-regulatory elements in the genome. TFs are multidomain proteins that arise evolutionarily through protein domain shuffling. The modular nature of TFs has led to the idea that specific modules of TFs can be re-designed to regulate desired gene(s) through protein engineering. Utilization of designer TFs for the control of metabolic pathways has emerged as an effective approach for metabolic engineering. We are interested in engineering the basic helix-loop-helix (bHLH, Myc-type) transcription factors. Using site-directed and saturation mutagenesis, in combination with efficient and high-throughput screening systems, we have identified and characterized several amino acid residues critical for higher transactivation activity of a Myc-like bHLH transcription factor involved in anthocyanin biosynthetic pathway in plants. Site-directed and saturation mutagenesis should be generally applicable to engineering of all TFs.

  20. Greetings from The International Association of Environmental Mutagenesis and Genomics Societies.

    PubMed

    Nohmi, Takehiko

    2015-01-01

    The International Association of Environmental Mutagenesis and Genomics Societies (IAEMGS) is an organization that promotes basic and applied research on environmental mutagenesis and genomics. In this article, as President of IAEMGS, I stress the important role of Genes and Environment to spread the voice of Asia to the international scientific community. Open access will support the journal in achieving this mission.

  1. Establishment of a Counter-selectable Markerless Mutagenesis System in Veillonella atypica

    PubMed Central

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-01-01

    Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella. PMID:25771833

  2. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  3. Environmental mutagenesis during the end-Permian ecological crisis.

    PubMed

    Visscher, Henk; Looy, Cindy V; Collinson, Margaret E; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H A; Kürschner, Wolfram M; Sephton, Mark A

    2004-08-31

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism.

  4. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  5. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

    PubMed Central

    Chong, Nikson Fatt-Ming

    2016-01-01

    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. PMID:27995143

  6. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  7. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  8. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  9. In vivo site-directed mutagenesis using oligonucleotides.

    PubMed

    Storici, F; Lewis, L K; Resnick, M A

    2001-08-01

    Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

  10. Structure-based design of combinatorial mutagenesis libraries.

    PubMed

    Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

    2015-05-01

    The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called "Structure-based Optimization of Combinatorial Mutagenesis" (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states.

  11. The HIV mutation browser: a resource for human immunodeficiency virus mutagenesis and polymorphism data.

    PubMed

    Davey, Norman E; Satagopam, Venkata P; Santiago-Mozos, Salvador; Villacorta-Martin, Carlos; Bharat, Tanmay A M; Schneider, Reinhard; Briggs, John A G

    2014-12-01

    Huge research effort has been invested over many years to determine the phenotypes of natural or artificial mutations in HIV proteins--interpretation of mutation phenotypes is an invaluable source of new knowledge. The results of this research effort are recorded in the scientific literature, but it is difficult for virologists to rapidly find it. Manually locating data on phenotypic variation within the approximately 270,000 available HIV-related research articles, or the further 1,500 articles that are published each month is a daunting task. Accordingly, the HIV research community would benefit from a resource cataloguing the available HIV mutation literature. We have applied computational text-mining techniques to parse and map mutagenesis and polymorphism information from the HIV literature, have enriched the data with ancillary information and have developed a public, web-based interface through which it can be intuitively explored: the HIV mutation browser. The current release of the HIV mutation browser describes the phenotypes of 7,608 unique mutations at 2,520 sites in the HIV proteome, resulting from the analysis of 120,899 papers. The mutation information for each protein is organised in a residue-centric manner and each residue is linked to the relevant experimental literature. The importance of HIV as a global health burden advocates extensive effort to maximise the efficiency of HIV research. The HIV mutation browser provides a valuable new resource for the research community. The HIV mutation browser is available at: http://hivmut.org.

  12. Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

    PubMed

    Xu, Zhong; Wang, Yemin; Chater, Keith F; Ou, Hong-Yu; Xu, H Howard; Deng, Zixin; Tao, Meifeng

    2017-03-15

    Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation

  13. Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

    PubMed

    Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

    2014-01-01

    Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

  14. Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop.

    PubMed

    Wu, S J; Koller, C N; Miller, D L; Bauer, L S; Dean, D H

    2000-05-12

    We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R(345)A,Y(350)F,Y(351)F) and A2 (R(345)A,DeltaY(350), DeltaY(351)), showed significantly improved toxicity against Tenebrio molitor (yellow mealworm). The mutant toxin A1 was also more potent against both Leptinotarsa decemlineata (Colorado potato beetle) and Chrysomela scripta (cottonwood leaf beetle), while A2 displayed enhanced toxicity only in L. decemlineata. Competitive binding assays of L. decemlineata brush border membrane vesicles (BBMV) revealed that binding affinities for the A1 and A2 mutant toxins were ca. 2.5-fold higher than for the wild-type Cry3 toxin. Similar binding assays with C. scripta BBMV revealed a ca. 5-fold lower dissociation rate for the A1 mutant as compared to that of Cry3A.

  15. Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species.

    PubMed

    Um, Hae Young; Chung, Eunsook; Lee, Jai-Heon; Lee, Seon-Woo

    2011-04-01

    Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.

  16. Radiation quality and mutagenesis in human lymphoblastoid cells.

    PubMed

    Liber, Howard L; Idate, Rupa; Warner, Christy; Bailey, Susan M

    2014-10-01

    , there was no clear evidence of a dose response for bystander mutagenesis, i.e., the MF plateaued. Interestingly, the magnitudes of the bystander MFs induced by different ion/energy combinations did vary, with bystander MFs ranging from 0.8 to 2.2× higher than the background. Furthermore, the nontargeted MFs appeared to reflect a mirror image of that for direct mutagenesis.

  17. Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis

    PubMed Central

    Idnurm, Alexander; Reedy, Jennifer L.; Nussbaum, Jesse C.; Heitman, Joseph

    2004-01-01

    Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their

  18. Genes Necessary for Bacterial Magnetite Biomineralization Identified by Transposon Mutagenesis

    NASA Astrophysics Data System (ADS)

    Nash, C. Z.; Komeili, A.; Newman, D. K.; Kirschvink, J. L.

    2004-12-01

    Magnetic bacteria synthesize nanoscale crystals of magnetite in intracellular, membrane-bounded organelles (magnetosomes). These crystals are preserved in the fossil record at least as far back as the late Neoproterozoic and have been tentatively identified in much older rocks (1). This fossil record may provide deep time calibration points for molecular evolution studies once the genes involved in biologically controlled magnetic mineralization (BCMM) are known. Further, a genetic and biochemical understanding of BCMM will give insight into the depositional environment and biogeochemical cycles in which magnetic bacteria play a role. The BCMM process is not well understood, though proteins have been identified from the magnetosome membrane and genetic manipulation and biochemical characterization of these proteins are underway. Most of the proteins currently thought to be involved are encoded within the mam cluster, a large cluster of genes whose products localize to the magnetosome membrane and are conserved among magnetic bacteria (2). In an effort to identify all of the genes necessary for bacterial BCMM, we undertook a transposon mutagenesis of Magnetospirillum magneticum AMB-1. Non-magnetic mutants (MNMs) were identified by growth in liquid culture followed by a magnetic assay. The insertion site of the transposon was identified two ways. First MNMs were screened with a PCR assay to determine if the transposon had inserted into the mam cluster. Second, the transposon was rescued from the mutant DNA and cloned for sequencing. The majority insertion sites are located within the mam cluster. Insertion sites also occur in operons which have not previously been suspected to be involved in magnetite biomineralization. None of the insertion sites have occurred within genes reported from previous transposon mutagenesis studies of AMB-1 (3, 4). Two of the non-mam cluster insertion sites occur in operons containing genes conserved particularly between MS-1 and MC-1. We

  19. Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

    PubMed Central

    Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

    2015-01-01

    The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

  20. Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis

    PubMed Central

    Amano, Yoshihiko

    2016-01-01

    Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5–9.0 and possessed high thermal stability up to 50°C with 80% of the residual activities compared with the stability seen at the pH range of 6.5–7.0 and temperatures of up to 40°C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases. PMID:26790148

  1. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

    SciTech Connect

    Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.; Farrington, Graham; Lugovskoy, Alexey; Walus, Lee; Eldredge, John; Capili, Allan; Mi, Sha; Graff, Christilyn; Garber, Ellen

    2010-11-15

    Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

  2. System-dependent regulations of colour-pattern development: a mutagenesis study of the pale grass blue butterfly.

    PubMed

    Iwata, Masaki; Hiyama, Atsuki; Otaki, Joji M

    2013-01-01

    Developmental studies on wing colour patterns have been performed in nymphalid butterflies, but efficient genetic manipulations, including mutagenesis, have not been well established. Here, we have performed mutagenesis experiments in a lycaenid butterfly, the pale grass blue Zizeeria maha, to produce colour-pattern mutants. We fed the P-generation larvae an artificial diet containing the mutagen ethyl methane sulfonate (EMS), and the F1- and F2-generation adults showed various aberrant colour patterns: dorsoventral transformation, anterioposterior background colouration gap, weak contrast, disarrangement of spots, reduction of the size of spots, loss of spots, fusion of spots, and ectopic spots. Among them, the disarrangement, reduction, and loss of spots were likely produced by the coordinated changes of many spots of a single wing around the discal spot in a system-dependent manner, demonstrating the existence of the central symmetry system. The present study revealed multiple genetic regulations for system-dependent and wing-wide colour-pattern determination in lycaenid butterflies.

  3. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis.

    PubMed

    Pepinsky, R Blake; Silvian, Laura; Berkowitz, Steven A; Farrington, Graham; Lugovskoy, Alexey; Walus, Lee; Eldredge, John; Capili, Allan; Mi, Sha; Graff, Christilyn; Garber, Ellen

    2010-05-01

    Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

  4. Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle

    PubMed Central

    Posthuma, Clara C.; Nedialkova, Danny D.; Zevenhoven-Dobbe, Jessika C.; Blokhuis, Jeroen H.; Gorbalenya, Alexander E.; Snijder, Eric J.

    2006-01-01

    The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined. PMID:16439522

  5. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

    PubMed Central

    Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

    2016-01-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  6. Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis

    PubMed Central

    Sherif, Mohammed; Waung, Debbie; Korbeci, Bihter; Mavisakalyan, Valentina; Flick, Robert; Brown, Greg; Abou-Zaid, Mamdouh; Yakunin, Alexander F; Master, Emma R

    2013-01-01

    Summary Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (± 0.8) s−1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half-life of over 10 h at 80°C and over 7 h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates. Funding Information Funding for this research was provided by the Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’ (ORF-RE-05-005), and the Natural Sciences and Engineering Research Council of Canada. PMID:23815400

  7. Radio frequency electromagnetic fields: cancer, mutagenesis, and genotoxicity.

    PubMed

    Heynick, Louis N; Johnston, Sheila A; Mason, Patrick A

    2003-01-01

    We present critiques of epidemiologic studies and experimental investigations, published mostly in peer-reviewed journals, on cancer and related effects from exposure to nonionizing electromagnetic fields in the nominal frequency range of 3 kHz to 300 GHz of interest to Subcommittee 4 (SC4) of the International Committee on Electromagnetic Safety (ICES). The major topics discussed are presented under the headings Epidemiologic and Other Findings on Human Exposure, Mammals Exposed In Vivo, Mammalian Live Tissues and Cell Preparations Exposed In Vitro, and Mutagenesis and Genotoxicity in Microorganisms and Fruit Flies. Under each major topic, we present minireviews of papers on various specific endpoints investigated. The section on Epidemiologic and Other Findings on Human Exposure is divided into two subsections, the first on possible carcinogenic effects of exposure from emitters not in physical contact with the populations studied, for example, transmitting antennas and other devices. Discussed in the second subsection are studies of postulated carcinogenic effects from use of mobile phones, with prominence given to brain tumors from use of cellular and cordless telephones in direct physical contact with an ear of each subject. In both subsections, some investigations yielded positive findings, others had negative findings, including papers directed toward experimentally verifying positive findings, and both were reported in a few instances. Further research on various important aspects may resolve such differences. Overall, however, the preponderance of published epidemiologic and experimental findings do not support the supposition that in vivo or in vitro exposures to such fields are carcinogenic.

  8. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

  9. ENU mutagenesis in mice identifies candidate genes for hypogonadism.

    PubMed

    Weiss, Jeffrey; Hurley, Lisa A; Harris, Rebecca M; Finlayson, Courtney; Tong, Minghan; Fisher, Lisa A; Moran, Jennifer L; Beier, David R; Mason, Christopher; Jameson, J Larry

    2012-06-01

    Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

  10. Combinatorial mutagenesis and selection to understand and improve yeast promoters.

    PubMed

    Berg, Laila; Strand, Trine Aakvik; Valla, Svein; Brautaset, Trygve

    2013-01-01

    Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible P AOX1 promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant P AOX1 promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the P AOX1 promoter DNA sequence. We demonstrate that this approach can be used to select for P AOX1 promoter variants with abolished glucose repression in large mutant libraries. We also selected P AOX1 promoter variants with elevated expression level under induced conditions. The properties of the selected P AOX1 promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

  11. A Synthetic Approach to Stop-Codon Scanning Mutagenesis

    PubMed Central

    Nie, Lihua; Lavinder, Jason J.; Sarkar, Mohosin; Stephany, Kimberly; Magliery, Thomas J.

    2011-01-01

    A general combinatorial mutagenesis strategy using common DMT-protected mononucleotide phosphoramidites and a single orthogonally-protected trinucleotide phosphoramidite (Fmoc-TAG) was developed to scan a gene with the TAG amber stop codon with complete synthetic control. In combination with stop-codon suppressors that insert natural (e.g., alanine) or unnatural (e.g., p-benzoylphenylalanine or Bpa) amino acids, a single DNA library can be used to incorporate different amino acids for diverse purposes. Here, we scanned TAG codons through part of the gene for a model four-helix bundle protein, Rop, which regulates the copy number of ColE1 plasmids. Alanine was incorporated into Rop for mapping its binding site using an in vivo activity screen, and subtle but important differences from in vitro gel-shift studies of Rop function are evident. As a test, Bpa was incorporated using a Phe14 amber mutant isolated from the scanning library. Surprisingly, Phe14Bpa Rop is weakly active, despite the critical role of Phe14 in Rop activity. Bpa is a photoaffinity label unnatural amino acid that can form covalent bonds with adjacent molecules upon UV irradiation. Irradiation of Phe14Bpa-Rop, which is a dimer in solution like wild-type Rop, results in covalent dimers, trimers and tetramers. This suggests that Phe14Bpa Rop weakly associates as a tetramer in solution and highlights the use of Bpa crosslinking as a means of trapping weak and transient interactions. PMID:21452871

  12. Insertional mutagenesis of preneoplastic astrocytes by Moloney murine leukemia virus.

    PubMed

    Afanasieva, T A; Pekarik, V; Grazia D'Angelo, M; Klein, M A; Voigtländer, T; Stocking, C; Aguzzi, A

    2001-04-01

    Retroviral infection can induce transcriptional activation of genes flanking the sites of proviral integration in target cells. Because integration is essentially random, this phenomenon can be exploited for random mutagenesis of the genome, and analysis of integration sites in tumors may identify potential oncogenes. Here we have investigated this strategy in the context of astrocytoma progression. Neuroectodermal explants from astrocytoma-prone GFAP-v-src transgenic mice were infected with the ecotropic Moloney murine leukemia virus (Mo-MuLV). In situ hybridization and FACS analysis indicated that astrocytes from E12.5-13.5 embryos were highly susceptible to retroviral infection and expressed viral RNA and proteins both in vitro and in vivo. In average 80% of neuroectodermal cells were infected in vitro with 9-14 proviral integrations per cell. Virus mobility assays confirmed that Mo-MuLV remained transcriptionally active and replicating in neuroectodermal primary cultures even after 45 days of cultivation. Proviral insertion sites were investigated by inverse long-range PCR. Analysis of a limited number of provirus flanking sequences in clones originated from in vitro infected GFAP-v-src neuroectodermal cells identified loci of possible relevance to tumorigenesis. Therefore, the approach described here might be suitable for acceleration of tumorigenesis in preneoplastic astrocytes. We expect this method to be useful for identifying genes involved in astrocytoma development/progression in animal models.

  13. Analysis of HIV-2 Vpx by modeling and insertional mutagenesis

    SciTech Connect

    Mahnke, Lisa A. . E-mail: lmahnke@im.wustl.edu; Belshan, Michael; Ratner, Lee . E-mail: lratner@im.wustl.edu

    2006-04-25

    Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.

  14. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  15. Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase

    PubMed Central

    Özgün, Gülşah P.; Ordu, Emel B.; Tütüncü, H. Esra; Yelboğa, Emrah; Sessions, Richard B.

    2016-01-01

    In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH. PMID:27847673

  16. Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization.

    PubMed

    Cheema, Pawanjit Singh; Singh, Satparkash; Kathiresan, S; Kumar, Ramesh; Bhanot, Vandna; Singh, Vijendra Pal

    2017-01-02

    Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG's and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at ∼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

  17. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  18. An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

    PubMed Central

    Fitzgerald, Timothy L.; Powell, Jonathan J.; Stiller, Jiri; Weese, Terri L.; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C. Lynne; Li, Zhongyi; Manners, John M.; Kazan, Kemal

    2015-01-01

    Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed. PMID:25719507

  19. Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase.

    PubMed Central

    Escobar, W A; Miller, J; Fink, A L

    1994-01-01

    Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding. PMID:7980417

  20. Molecular Mechanisms for High Hydrostatic Pressure-Induced Wing Mutagenesis in Drosophila melanogaster.

    PubMed

    Wang, Hua; Wang, Kai; Xiao, Guanjun; Ma, Junfeng; Wang, Bingying; Shen, Sile; Fu, Xueqi; Zou, Guangtian; Zou, Bo

    2015-10-08

    Although High hydrostatic pressure (HHP) as an important physical and chemical tool has been increasingly applied to research of organism, the response mechanisms of organism to HHP have not been elucidated clearly thus far. To identify mutagenic mechanisms of HHP on organisms, here, we treated Drosophila melanogaster (D. melanogaster) eggs with HHP. Approximately 75% of the surviving flies showed significant morphological abnormalities from the egg to the adult stages compared with control flies (p < 0.05). Some eggs displayed abnormal chorionic appendages, some larvae were large and red, and some adult flies showed wing abnormalities. Abnormal wing phenotypes of D. melanogaster induced by HHP were used to investigate the mutagenic mechanisms of HHP on organism. Thus 285 differentially expressed genes associated with wing mutations were identified using Affymetrix Drosophila Genome Array 2.0 and verified with RT-PCR. We also compared wing development-related central genes in the mutant flies with control flies using DNA sequencing to show two point mutations in the vestigial (vg) gene. This study revealed the mutagenic mechanisms of HHP-induced mutagenesis in D. melanogaster and provided a new model for the study of evolution on organisms.

  1. Assigning biological functions to rice genes by genome annotation, expression analysis and mutagenesis.

    PubMed

    Jiang, Shu-Ye; Ramachandran, Srinivasan

    2010-12-01

    Rice is the first cereal genome to be completely sequenced. Since the completion of its genome sequencing, considerable progress has been made in multiple areas including the whole genome annotation, gene expression profiling, mutant collection, etc. Here, we summarize the current status of rice genome annotation and review the methodology of assigning biological functions to hundreds of thousands of rice genes as well as discuss the major limitations and the future perspective in rice functional genomics. Available data analysis shows that the rice genome encodes around 32,000 protein-coding genes. Expression analysis revealed at least 31,000 genes with expression evidence from full-length cDNA/EST collection or other transcript profiling. In addition, we have summarized various strategies to generate mutant population including natural, physical, chemical, T-DNA, transposon/retrotransposon or gene silencing based mutagenesis. Currently, more than 1 million of mutants have been generated and 27,551 of them have their flanking sequence tags. To assign biological functions to hundreds of thousands of rice genes, global co-operations are required, various genetic resources should be more easily accessible and diverse data from transcriptomics, proteomics, epigenetics, comparative genomics and bioinformatics should be integrated to better understand the functions of these genes and their regulatory mechanisms.

  2. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

  3. Enhancement of 1,3-Dihydroxyacetone Production from Gluconobacter oxydans by Combined Mutagenesis.

    PubMed

    Lin, Xi; Liu, Sha; Xie, Guangrong; Chen, Jing; Li, Penghua; Chen, Jianhua

    2016-11-28

    Wild strain L-6 was subjected to combined mutagenesis, including UV irradiation, atmospheric and room temperature plasma, and ion beam implantation, to increase the yield of 1,3-dihydroxyacetone (DHA). With application of a high-throughput screening method, mutant Gluconobacter oxydans I-2-239 with a DHA productivity of 103.5 g/l in flask-shake fermentation was finally obtained with the starting glycerol concentration of 120 g/l, which was 115.7% higher than the wild strain. The cultivation time also decreased from 54 h to 36 h. Compared with the wild strain, a dramatic increase in enzyme activity was observed for the mutant strain, although the increase in biomass was limited. DNA and amino acid sequence alignment revealed 11 nucleotide substitutions and 10 amino acid substitutions between the sldAB of strains L-6 and I-2-239. Simulation of the 3-D structure and prediction of active site residues and PQQ binding site residues suggested that these mutations were mainly related to PQQ binding, which was speculated to be favorable for the catalyzing capacity of glycerol dehydrogenase. RT-qPCR assay indicated that the transcription levels of sldA and sldB in the mutant strain were respectively 4.8-fold and 5.4-fold higher than that in the wild strain, suggesting another possible reason for the increased DHA productivity of the mutant strain.

  4. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    PubMed

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

  5. Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.

    PubMed

    Marotti, K R; Tomich, C S

    1989-01-01

    A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

  6. Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

    PubMed Central

    Ortega-Prieto, Ana M.; Sheldon, Julie; Grande-Pérez, Ana; Tejero, Héctor; Gregori, Josep; Quer, Josep; Esteban, Juan I.; Domingo, Esteban; Perales, Celia

    2013-01-01

    Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

  7. [Dot1 and Set2 Histone Methylases Control the Spontaneous and UV-Induced Mutagenesis Levels in the Saccharomyces cerevisiae Yeasts].

    PubMed

    Kozhina, T N; Evstiukhina, T A; Peshekhonov, V T; Chernenkov, A Yu; Korolev, V G

    2016-03-01

    In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of hi- stone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-in- duced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the sponta- neous mutagenesis rate in both single and d ouble mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the ho- mologous-recombination-based and the postreplicative DNA repair.

  8. Role of mouse germ-cell mutagenesis in understanding genetic risk and in generating mutations that are prime tools for studies in modern biology.

    PubMed

    Russell, L B

    1994-01-01

    Highlights are presented on (1) the role mouse germ-cell mutagenesis has played in assessing the genetic harm from radiations and chemicals, and (2) the contributions to the field of modern biology that are being made by the products of this research--the propagated mutations. Among the numerous findings in radiation mutagenesis were the humped dose-effect curve for spermatogonial stem cells, the major differences between the sexes and between germ-cell stages of each sex in both yield and nature of mutations, the dose-rate effect, which provided the first evidence for repair of mutational (or premutational) damage, the augmenting effect of certain regimes of dose fractionation, and many others. Chemical mutagenesis studies that followed revealed at least three patterns of mutation yield and demonstrated that germ-cell stage--much more than the nature of the chemical--governs the nature of the DNA lesions induced. Two "supermutagens," one for intragenic mutations and one for deletions and other rearrangements, have become very useful in the manufacture of mutations for specific purposes. The mutations propagated from radiation- and chemical-mutagenesis experiments are providing prime resources for basic studies in genome organization, gene structure, and function. DNA lesions that involve specific loci have made possible increasingly detailed characterization of extensive deletion complexes that facilitate high-intensity physical and functional mapping within them. Numerous loci associated with interesting developmental anomalies have been identified and have become accessible to positional cloning. Several of the genes accessed with the aid of induced mutations (deletions, other rearrangements, and point mutations) are furnishing prime reagents for elucidating human disease conditions.

  9. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity.

    PubMed

    Wu, Chuanfeng; Dunbar, Cynthia E

    2011-12-01

    Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application.

  10. Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice.

    PubMed

    Gallego-Llamas, Jabier; Timms, Andrew E; Pitstick, Rose; Peters, Janet; Carlson, George A; Beier, David R

    2016-01-01

    ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1-2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest.

  11. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  12. Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing

    PubMed Central

    Jee, Justin; Rasouly, Aviram; Shamovsky, Ilya; Akivis, Yonatan; Steinman, Susan; Mishra, Bud; Nudler, Evgeny

    2016-01-01

    In 1943, Luria and Delbrück used a phage resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). We devise a method, Maximum-Depth Sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in E. coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress; transcription-replication conflicts; and in the case of fluoroquinolones, direct damage to DNA. PMID:27338792

  13. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    NASA Technical Reports Server (NTRS)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  14. Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice

    PubMed Central

    Pitstick, Rose; Peters, Janet; Carlson, George A.

    2016-01-01

    ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1–2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest. PMID:27441645

  15. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-01-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and [gamma]-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by [gamma]-rays, [alpha]-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than [gamma]-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate [gamma]-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed.

  16. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-02-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and {gamma}-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by {gamma}-rays, {alpha}-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than {gamma}-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate {gamma}-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed.

  17. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  18. Generation of Enterobacter sp. YSU auxotrophs using transposon mutagenesis.

    PubMed

    Caguiat, Jonathan James

    2014-10-31

    Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host's genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a

  19. Involvement of 5-methylcytosine in sunlight-induced mutagenesis.

    PubMed

    You, Y H; Li, C; Pfeifer, G P

    1999-10-29

    In human skin cancers, more than 30 % of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. In Xorder to define the contribution of 5-methylcytosine to sunlight-induced mutations, we have used mouse fibroblasts containing the CpG-methylated lacI transgene as a mutational target. We sequenced 182 UVC (254 nm UV)-induced mutations and 170 mutations induced by a solar UV simulator, along with 75 mutations in untreated cells. Only a few of the mutations in untreated cells were transitions at dipyrimidines, but more than 95% of the UVC and solar irradiation-induced mutations were targeted to dipyrimidine sites, the majority being transitions. After UVC irradiation, 6% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only 2.2% of all mutations were transitions within this sequence context. However, 24% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Two sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form the highest levels of cyclobutane pyrimidine dimers after irradiation with sunlight but not with UVC. The data indicate that dipyrimidines that contain 5-methylcytosine are preferential targets for sunlight-induced mutagenesis in cultured mammalian cells, thus explaining the large proportion of p53 mutations at such sites in skin tumors in vivo.

  20. Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

    PubMed Central

    Pandyarajan, Vijay; Phillips, Nelson B.; Cox, Gabriela P.; Yang, Yanwu; Whittaker, Jonathan; Ismail-Beigi, Faramarz; Weiss, Michael A.

    2014-01-01

    Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed LysB28, ProB29-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ±0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. PMID:24993826

  1. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  2. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  3. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data

    PubMed Central

    Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C.; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J.; Aghera, Nilesh; Varadarajan, Raghavan

    2016-01-01

    Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. PMID:27563054

  4. Orchestrating Proactive and Reactive Mechanisms for Filtering Distracting Information: Brain-Behavior Relationships Revealed by a Mixed-Design fMRI Study.

    PubMed

    Marini, Francesco; Demeter, Elise; Roberts, Kenneth C; Chelazzi, Leonardo; Woldorff, Marty G

    2016-01-20

    Given the information overload often imparted to human cognitive-processing systems, suppression of irrelevant and distracting information is essential for successful behavior. Using a hybrid block/event-related fMRI design, we characterized proactive and reactive brain mechanisms for filtering distracting stimuli. Participants performed a flanker task, discriminating the direction of a target arrow in the presence versus absence of congruent or incongruent flanking distracting arrows during either Pure blocks (distracters always absent) or Mixed blocks (distracters on 80% of trials). Each Mixed block had either 20% or 60% incongruent trials. Activations in the dorsal frontoparietal attention network during Mixed versus Pure blocks evidenced proactive (blockwise) recruitment of a distraction-filtering mechanism. Sustained activations in right middle frontal gyrus during 60% Incongruent blocks correlated positively with behavioral indices of distraction-filtering (slowing when distracters might occur) and negatively with distraction-related behavioral costs (incongruent vs congruent trials), suggesting a role in coordinating proactive filtering of potential distracters. Event-related analyses showed that incongruent trials elicited greater reactive activations in 20% (vs 60%) Incongruent blocks for counteracting distraction and conflict, including in the insula and anterior cingulate. Context-related effects in occipitoparietal cortex consisted of greater target-evoked activations for distracter-absent trials (central-target-only) in Mixed versus Pure blocks, suggesting enhanced attentional engagement. Functional-localizer analyses in V1/V2/V3 revealed less distracter-processing activity in 60% (vs 20%) Incongruent blocks, presumably reflecting tonic suppression by proactive filtering mechanisms. These results delineate brain mechanisms underlying proactive and reactive filtering of distraction and conflict, and how they are orchestrated depending on distraction

  5. Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor.

    PubMed

    Goffinont, Stephane; Villette, Sandrine; Spotheim-Maurizot, Melanie

    2012-06-01

    The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.

  6. Size at the onset of maturity (SOM) revealed in length-weight relationships of brackish amphipods and isopods: An information theory approach

    NASA Astrophysics Data System (ADS)

    Longo, Emanuela; Mancinelli, Giorgio

    2014-01-01

    In amphipods and other small-sized crustaceans, allometric relationships are conventionally analysed by fitting the standard model Y = a·Xb (X and Y are, e.g., body length and weight, respectively) whose scaling exponent b is assumed to be constant. However, breakpoints in allometric relationships have long been documented in large-sized crustaceans, ultimately determined by ontogenetic, abrupt variations in the value of b. Here, the existence of breakpoints in length-weight relationships was investigated in four amphipod (i.e., Gammarus aequicauda, Gammarus insensibilis, Microdeutopus gryllotalpa, and Dexamine spinosa) and three isopod species (i.e., Lekanesphaera hookeri, Sphaeroma serratum, and Cymodoce truncata) from three Mediterranean lagoons. The power of two candidate linear models fitted to log10-transformed data - a simple model assuming a constant exponent b and a segmented model assuming b to vary after a breakpoint - was compared using a parsimonious selection strategy based on the Akaike information criterion. The segmented model with a breakpoint provided the most accurate fitting of length-weight data in the majority of the species analysed; non-conclusive results were obtained only for D. spinosa and C. truncata, of which a limited number of specimens was examined. Model parameters were consistent for amphipod and isopod species collected across the three different habitats; the generality of the results was further supported by a literature search confirming that the identified breakpoints corresponded with ontogenetic discontinuities related with sexual maturation in all the species investigated. In this study, segmented regression models were revealed to provide a statistically accurate and biologically meaningful description of length-weight relationships of common amphipod and isopod species. The methodological limitations of the approach are considered, while the practical implications for secondary production estimates are discussed.

  7. Revealing Rembrandt

    PubMed Central

    Parker, Andrew J.

    2014-01-01

    The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI). Our results emphasized the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt's portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings. PMID:24795552

  8. Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

    PubMed Central

    2010-01-01

    Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID

  9. Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY.

    PubMed

    Sáez-Jiménez, Verónica; Baratto, Maria Camilla; Pogni, Rebecca; Rencoret, Jorge; Gutiérrez, Ana; Santos, José Ignacio; Martínez, Angel T; Ruiz-Dueñas, Francisco Javier

    2015-09-18

    Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn(2+), and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.

  10. Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.

    PubMed

    Tang, Lixia; Zheng, Kai; Liu, Yu; Zheng, Huayu; Wang, Hu; Song, Chunlei; Zhou, Hong

    2013-01-01

    Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg(2+) , and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.

  11. 2012 Gordon Research Conference on Mutagenesis - Formal Schedule and Speaker/Poster Program

    SciTech Connect

    Demple, Bruce

    2012-08-24

    The delicate balance among cellular pathways that control mutagenic changes in DNA will be the focus of the 2012 Mutagenesis Gordon Research Conference. Mutagenesis is essential for evolution, while genetic stability maintains cellular functions in all organisms from microbes to metazoans. Different systems handle DNA lesions at various times of the cell cycle and in different places within the nucleus, and inappropriate actions can lead to mutations. While mutation in humans is closely linked to disease, notably cancers, mutational systems can also be beneficial. The conference will highlight topics of beneficial mutagenesis, including full establishment of the immune system, cell survival mechanisms, and evolution and adaptation in microbial systems. Equal prominence will be given to detrimental mutation processes, especially those involved in driving cancer, neurological diseases, premature aging, and other threats to human health. Provisional session titles include Branching Pathways in Mutagenesis; Oxidative Stress and Endogenous DNA Damage; DNA Maintenance Pathways; Recombination, Good and Bad; Problematic DNA Structures; Localized Mutagenesis; Hypermutation in the Microbial World; and Mutation and Disease.

  12. Assessment of the mutagenic potential of ethanol auto engine exhaust gases by the Salmonella typhimurium microsomal mutagenesis assay, using a direct exposure method

    SciTech Connect

    Lotfi, C.F.; Brentani, M.M.; Boehm, G.M. )

    1990-08-01

    The mutagenic activity of the new Brazilian fuel, ethanol, was determined by employing the Salmonella typhimurium microsomal mutagenesis assay (TA97, TA98, TA100, TA102, and TA104) and a direct exposure method. This methodology was first used to determine the mutagenic activity of gasoline, revealing mutagenic activity of base-pair substitution without any need for metabolic activation, indicating the presence of direct-action mutagens. Experiments with ethanol suggest an indirect mutagenic activity of the oxidant type. The exposure system was considered suitable for future studies of gaseous mixtures.

  13. Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

    PubMed

    Shaw, Craig D; Storek, Michael J; Young, Kendra A; Kovacs, James M; Thurman, Joshua M; Holers, V Michael; Hannan, Jonathan P

    2010-12-10

    The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is

  14. Thiostrepton Variants Containing a Contracted Quinaldic Acid Macrocycle Result from Mutagenesis of the Second Residue

    PubMed Central

    Zhang, Feifei; Li, Chaoxuan

    2016-01-01

    The thiopeptides are a family of ribosomally synthesized and posttranslationally modified peptide metabolites, and the vast majority of thiopeptides characterized to date possess one highly modified macrocycle. A few members, including thiostrepton A, harbor a second macrocycle that incorporates a quinaldic acid moiety and the four N-terminal residues of the peptide. The antibacterial properties of thiostrepton A are well established, and its recently discovered ability to inhibit the proteasome has additional implications for the development of antimalarial and anticancer therapeutics. We have conducted the saturation mutagenesis of Ala2 in the precursor peptide, TsrA, to examine which variants can be transformed into a mature thiostrepton analogue. Although the thiostrepton biosynthetic system is somewhat restrictive towards substitutions at the second residue, eight thiostrepton Ala2 analogues were isolated. The TsrA Ala2Ile and Ala2Val variants were largely channeled through an alternate processing pathway wherein the first residue of the core peptide, Ile1, is removed and the resulting thiostrepton analogues bear quinaldic acid macrocycles abridged by one residue. This is the first report revealing that quinaldic acid loop size is amenable to alteration during the course of thiostrepton biosynthesis. Both the antibacterial and proteasome inhibitory properties of the thiostrepton Ala2 analogues were examined. While the identity of the residue at the second position of the core peptide influences thiostrepton biosynthesis, our report suggests it may not be crucial for antibacterial and proteasome inhibitory properties of the full-length variants. In contrast, the contracted quinaldic acid loop can, to differing degrees, affect both types of biological activity. PMID:26630475

  15. First Streptococcus pyogenes signature-tagged mutagenesis screen identifies novel virulence determinants.

    PubMed

    Kizy, Anne E; Neely, Melody N

    2009-05-01

    The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.

  16. In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome

    PubMed Central

    Todt, Daniel; Gisa, Anett; Radonic, Aleksandar; Nitsche, Andreas; Behrendt, Patrick; Suneetha, Pothakamuri Venkata; Pischke, Sven; Bremer, Birgit; Brown, Richard J P; Manns, Michael P; Cornberg, Markus; Bock, C Thomas; Steinmann, Eike; Wedemeyer, Heiner

    2016-01-01

    Objective Hepatitis E virus (HEV) infection can take chronic courses in immunocompromised patients potentially leading to liver cirrhosis and liver failure. Ribavirin (RBV) is currently the only treatment option for many patients, but treatment failure can occur which has been associated with the appearance of a distinct HEV polymerase mutant (G1634R). Here, we performed a detailed analysis of HEV viral intrahost evolution during chronic hepatitis E infections. Design Illumina deep sequencing was performed for the detection of intrahost variation in the HEV genome of chronically infected patients. Novel polymerase mutants were investigated in vitro using state-of-the-art HEV cell culture models. Results Together, these data revealed that (1) viral diversity differed markedly between patients but did not show major intraindividual short-term variations in untreated patients with chronic hepatitis E, (2) RBV therapy was associated with an increase in viral heterogeneity which was reversible when treatment was stopped, (3) the G1634R mutant was detectable as a minor population prior to therapy in patients who subsequently failed to achieve a sustained virological response to RBV therapy and (4) in addition to G1634R further dominant variants in the polymerase region emerged, impacting HEV replication efficiency in vitro. Conclusions In summary, this first investigation of intrahost HEV population evolution indicates that RBV causes HEV mutagenesis in treated patients and that an emergence of distinct mutants within the viral population occurs during RBV therapy. We also suggest that next-generation sequencing could be useful to guide personalised antiviral strategies. PMID:27222534

  17. Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

    PubMed

    Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

    2016-01-01

    Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations.

  18. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool.

    PubMed

    Zhang, Xue; Zhang, Xiao-Fei; Li, He-Ping; Wang, Li-Yan; Zhang, Chong; Xing, Xin-Hui; Bao, Cheng-Yu

    2014-06-01

    Developing rapid and diverse microbial mutation tool is of importance to strain modification. In this review, a new mutagenesis method for microbial mutation breeding using the radio-frequency atmospheric-pressure glow discharge (RF APGD) plasma jets is summarized. Based on the experimental study, the helium RF APGD plasma jet has been found to be able to change the DNA sequences significantly, indicating that the RF APGD plasma jet would be a powerful tool for the microbial mutagenesis with its outstanding features, such as the low and controllable gas temperatures, abundant chemically reactive species, rapid mutation, high operation flexibility, etc. Then, with the RF APGD plasma generator as the core component, a mutation machine named as atmospheric and room temperature plasma (ARTP) mutation system has been developed and successfully employed for the mutation breeding of more than 40 kinds of microorganisms including bacteria, fungi, and microalgae. Finally, the prospect of the ARTP mutagenesis is discussed.

  19. Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus.

    PubMed

    Godfroy, Olivier; Peters, Akira F; Coelho, Susana M; Cock, J Mark

    2015-12-01

    Ectocarpus has emerged as a model organism for the brown algae and a broad range of genetic and genomic resources are being generated for this species. The aim of the work presented here was to evaluate two mutagenesis protocols based on ultraviolet irradiation and ethyl methanesulphonate treatment using genome resequencing to measure the number, type and distribution of mutations generated by the two methods. Ultraviolet irradiation generated a greater number of genetic lesions than ethyl methanesulphonate treatment, with more than 400 mutations being detected in the genome of the mutagenised individual. This study therefore confirms that the ultraviolet mutagenesis protocol is suitable for approaches that require a high density of mutations, such as saturation mutagenesis or Targeting Induced Local Lesions in Genomes (TILLING).

  20. Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

    PubMed Central

    Cummins, Joanne; Gahan, Cormac G.M.

    2012-01-01

    Signature tagged mutagenesis is a genetic approach that was developed to identify novel bacterial virulence factors. It is a negative selection method in which unique identification tags allow analysis of pools of mutants in mixed populations. The approach is particularly well suited to functional genetic analysis of the gastrointestinal phase of infection in foodborne pathogens and has the capacity to guide the development of novel vaccines and therapeutics. In this review we outline the technical principles underpinning signature-tagged mutagenesis as well as novel sequencing-based approaches for transposon mutant identification such as TraDIS (transposon directed insertion-site sequencing). We also provide an analysis of screens that have been performed in gastrointestinal pathogens which are a global health concern (Escherichia coli, Listeria monocytogenes, Helicobacter pylori, Vibrio cholerae and Salmonella enterica). The identification of key virulence loci through the use of signature tagged mutagenesis in mice and relevant larger animal models is discussed. PMID:22555467

  1. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

    PubMed Central

    Varshney, Gaurav Kumar

    2014-01-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  2. Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait

    PubMed Central

    Abt, Tom Den; Souffriau, Ben; Foulquié-Moreno, Maria R.; Duitama, Jorge; Thevelein, Johan M.

    2016-01-01

    Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to

  3. In silico functional dissection of saturation mutagenesis: Interpreting the relationship between phenotypes and changes in protein stability, interactions and activity

    PubMed Central

    Pires, Douglas E. V.; Chen, Jing; Blundell, Tom L.; Ascher, David B.

    2016-01-01

    Despite interest in associating polymorphisms with clinical or experimental phenotypes, functional interpretation of mutation data has lagged behind generation of data from modern high-throughput techniques and the accurate prediction of the molecular impact of a mutation remains a non-trivial task. We present here an integrated knowledge-driven computational workflow designed to evaluate the effects of experimental and disease missense mutations on protein structure and interactions. We exemplify its application with analyses of saturation mutagenesis of DBR1 and Gal4 and show that the experimental phenotypes for over 80% of the mutations correlate well with predicted effects of mutations on protein stability and RNA binding affinity. We also show that analysis of mutations in VHL using our workflow provides valuable insights into the effects of mutations, and their links to the risk of developing renal carcinoma. Taken together the analyses of the three examples demonstrate that structural bioinformatics tools, when applied in a systematic, integrated way, can rapidly analyse a given system to provide a powerful approach for predicting structural and functional effects of thousands of mutations in order to reveal molecular mechanisms leading to a phenotype. Missense or non-synonymous mutations are nucleotide substitutions that alter the amino acid sequence of a protein. Their effects can range from modifying transcription, translation, processing and splicing, localization, changing stability of the protein, altering its dynamics or interactions with other proteins, nucleic acids and ligands, including small molecules and metal ions. The advent of high-throughput techniques including sequencing and saturation mutagenesis has provided large amounts of phenotypic data linked to mutations. However, one of the hurdles has been understanding and quantifying the effects of a particular mutation, and how they translate into a given phenotype. One approach to overcome

  4. In silico functional dissection of saturation mutagenesis: Interpreting the relationship between phenotypes and changes in protein stability, interactions and activity.

    PubMed

    Pires, Douglas E V; Chen, Jing; Blundell, Tom L; Ascher, David B

    2016-01-22

    Despite interest in associating polymorphisms with clinical or experimental phenotypes, functional interpretation of mutation data has lagged behind generation of data from modern high-throughput techniques and the accurate prediction of the molecular impact of a mutation remains a non-trivial task. We present here an integrated knowledge-driven computational workflow designed to evaluate the effects of experimental and disease missense mutations on protein structure and interactions. We exemplify its application with analyses of saturation mutagenesis of DBR1 and Gal4 and show that the experimental phenotypes for over 80% of the mutations correlate well with predicted effects of mutations on protein stability and RNA binding affinity. We also show that analysis of mutations in VHL using our workflow provides valuable insights into the effects of mutations, and their links to the risk of developing renal carcinoma. Taken together the analyses of the three examples demonstrate that structural bioinformatics tools, when applied in a systematic, integrated way, can rapidly analyse a given system to provide a powerful approach for predicting structural and functional effects of thousands of mutations in order to reveal molecular mechanisms leading to a phenotype. Missense or non-synonymous mutations are nucleotide substitutions that alter the amino acid sequence of a protein. Their effects can range from modifying transcription, translation, processing and splicing, localization, changing stability of the protein, altering its dynamics or interactions with other proteins, nucleic acids and ligands, including small molecules and metal ions. The advent of high-throughput techniques including sequencing and saturation mutagenesis has provided large amounts of phenotypic data linked to mutations. However, one of the hurdles has been understanding and quantifying the effects of a particular mutation, and how they translate into a given phenotype. One approach to overcome

  5. Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait.

    PubMed

    Abt, Tom Den; Souffriau, Ben; Foulquié-Moreno, Maria R; Duitama, Jorge; Thevelein, Johan M

    2016-03-18

    Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to

  6. Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

    PubMed Central

    Ashman, C R; Davidson, R L

    1981-01-01

    The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools. PMID:6965099

  7. Endonuclease IV and exonuclease III are involved in the repair and mutagenesis of DNA lesions induced by UVB in Escherichia coli.

    PubMed

    Souza, L L; Eduardo, I R; Pádula, M; Leitão, A C

    2006-03-01

    Exonuclease III (Exo III) and endonuclease IV (Endo IV) play a critical role in the base excision repair (BER) of Escherichia coli. Both are endowed with AP endonucleolytic activity, cleaving the 5' phosphodiester bond adjacent to spontaneous or induced abasic sites in DNA. Although mutants defective in Exo III (xthA) are usually hypersensitive to oxidative agents such as hydrogen peroxide, near-UV-light and X-rays, mutants defective in Endo IV (nfo) are not as sensitive as the xthA strain. To further investigate the roles of these AP endonucleases in DNA repair, we evaluated the sensitivity and mutagenesis of xthA and nfo strains after UVB and compared with UVC light. Our results revealed that xthA but not nfo strain was hypersensitive to UVB. The use of Fe(+2) ion chelator (dipyridyl), prior to irradiation, completely protected the xthA mutant against UVB lethal lesions, suggesting the generation of toxic oxidative lesions mediated by transition metal reactions. The nfo strain displayed increased UVB-induced mutagenesis, which was significantly suppressed by pre-treatment with dipyridyl. Although xthA strain did not display increased mutagenesis after UVC and UVB treatments, this phenotype was not related to xthA mutation, but rather to an unknown secondary mutation specifying an antimutator phenotype. After UVB irradiation, the base substitution spectra of nfo strain revealed a bias towards AT-->GC transitions and GC-->CG transversions, which were also suppressed by previous treatment with the iron chelator. Overall, on the basis of the differential sensitivities and mutational spectra displayed after UVC and UVB treatments, we propose a role for Endo IV and Exo III to counteract DNA damage induced by the oxidative counterpart of UVB in E.coli.

  8. The application of hepatic P450 reductase null gpt delta mice in studying the role of hepatic P450 in genotoxic carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mutagenesis.

    PubMed

    Luan, Yang; Xing, Guozhen; Qi, Xinming; Wu, Mengjun; Li, Chenggang; Yao, Jun; Gong, Likun; Nohmi, Takehiko; Gu, Jun; Zhou, Wanhong; Zheng, Saijing; Ren, Jin

    2012-11-01

    The cytochrome P450 (P450 or CYP) is involved in both detoxification and metabolic activation of many carcinogens. In order to identify the role of hepatic P450 in the mutagenesis of genotoxic carcinogens, we generated a novel hepatic P450 reductase null (HRN) gpt delta mouse model, which lacks functional hepatic P450 on a gpt delta mouse background. In this study, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was used to treat HRN gpt delta mice and control littermates. Gene mutations in the liver and lungs were detected, and mutation spectra were analyzed. Pharmacokinetic analyses were performed, and tissue levels of NNK and metabolite were determined. NNK-induced mutant frequencies (MFs) were equivalent to spontaneous MFs in the liver, but increased more than 3 times in the lungs of HRN gpt delta mice compared to control mice. NNK-induced mutation spectra showed no difference between HRN gpt delta mice and control littermates. Toxicokinetic studies revealed reduced clearance of NNK with elevated tissue concentrations in HRN gpt delta mice. To our knowledge, these are the first data demonstrating that NNK cannot induce mutagenesis in the liver without P450 metabolic activation, but can induce mutagenesis in lungs by a hepatic P450-independent mechanism. Moreover, our data show that hepatic P450 plays a major role in the systemic clearance of NNK, thereby protecting the lungs against NNK-induced mutagenesis. Our model will be useful in establishing the role of hepatic versus extrahepatic P450-mediated mutagenesis, and the relative contributions of P450 compared to other biotransformation enzymes in the genotoxic carcinogens' activation.

  9. Metabolic effects of CYP2A6 and CYP2A13 on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced gene mutation-A mammalian cell-based mutagenesis approach

    SciTech Connect

    Chiang, Huai-chih; Wang, Chin-Ying; Lee, Hui-Ling; Tsou, Tsui-Chun

    2011-06-01

    Both cytochrome P450 2A6 (CYP2A6) and cytochrome P450 2A13 (CYP2A13) are involved in metabolic activation of tobacco-specific nitrosamines and may play important roles in cigarette smoking-induced lung cancer. Unlike CYP2A6, effects of CYP2A13 on the tobacco-specific nitrosamine-induced mutagenesis in lung cells remain unclear. This study uses a supF mutagenesis assay to examine the relative effects of CYP2A6 and CYP2A13 on metabolic activation of a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its resulting mutagenesis in human lung cells. A recombinant adenovirus-mediated CYP2A6/CYP2A13 expression system was established to specifically address the relative effects of these two CYPs. Mutagenesis results revealed that both CYP2A6 and CYP2A13 significantly enhanced the NNK-induced supF mutation and that the mutagenic effect of CYP2A13 was markedly higher than that of CYP2A6. Analysis of NNK metabolism indicated that {>=} 70% of NNK was detoxified to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), either with or without CYP2A6/CYP2A13 expression. Both CYP2A6 and CYP2A13 significantly enhanced the {alpha}-hydroxylation of NNK; and the {alpha}-hydroxylation activity of CYP2A13 was significantly higher than that of CYP2A6. Analysis of the NNK-related DNA adduct formation indicated that, in the presence of CYP2A13, NNK treatments caused marked increases in O{sup 6}-methylguanine (O{sup 6}-MeG). The present results provide the first direct in vitro evidence demonstrating the predominant roles of CYP2A13 in NNK-induced mutagenesis, possibly via metabolic activation of NNK {alpha}-hydroxylation.

  10. Use of the Photoactic Ability of a Bacterium to Teach the Genetic Principles of Random Mutagenesis & Mutant Screening

    ERIC Educational Resources Information Center

    Din, Neena; Bird, Terry H.; Berleman, James E.

    2007-01-01

    In this article, the authors present a laboratory activity that relies on the use of a very versatile bacterial system to introduce the concept of how mutagenesis can be used for molecular and genetic analysis of living organisms. They have used the techniques of random mutagenesis and selection/screening to obtain strains of the organism "R.…

  11. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol were produced from Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 using UV-C mutagenesis. Random UV-C mutagenesis potentially produces large numbers of mutations broadly and uniformly over the whole genome to genera...

  12. Comparative mutagenesis of human cells in vivo and in vitro

    SciTech Connect

    Thilly, W.G.

    1990-01-01

    Our goal is to develop the tools of mutational spectrometry in order to discover the cause(s) of genetic change in somatic and germinal cells in humans. Our study of the spectrum of point mutations in human mitochrondrial DNA sequences has revealed that there are multiple point mutation hotspots in each of four separate sequences in the mitochrondrial genome. These spectra were revealed by a combination of high fidelity PCR (modified T{sub 7} polymerase) and denaturing gradient gel electrophoresis which has a limit of detection of about 10{sup {minus}3}. There appear to be identical hotspot mutations in both cultured B cell and fresh human blood T cell samples.

  13. Information resources and the correlation of response patterns between biological end points

    SciTech Connect

    Malling, H.V.; Wassom, J.S.

    1990-12-31

    This paper focuses on the analysis of information for mutagenesis, a biological end point that is important in the overall process of assessing possible adverse health effects from chemical exposure. 17 refs.

  14. Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu.

    PubMed

    Tran, Thuy T; Mamo, Gashaw; Búxo, Laura; Le, Nhi N; Gaber, Yasser; Mattiasson, Bo; Hatti-Kaul, Rajni

    2011-07-10

    Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.

  15. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

    PubMed Central

    Ng, Ashley Shu Mei; Ingham, Philip W.

    2016-01-01

    CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. PMID:27832146

  16. Crystal Structures of Glycosyltransferase UGT78G1 Reveal the Molecular Basis for Glycosylation and Deglycosylation of (Iso)flavonoids

    SciTech Connect

    Modolo, Luzia V.; Li, Lenong; Pan, Haiyun; Blount, Jack W.; Dixon, Richard A.; Wang, Xiaoqiang

    2010-09-21

    The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 {angstrom} resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides a basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.

  17. Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant, opaque-2. Using gamma irradiation, we created opaque QPM variants to identify opaque-2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize functional genomics tool. A K0326...

  18. Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate mutagenesis of two G. hirsutum breeding lines, TAM 94L...

  19. Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

    PubMed

    Tingting, Liu; Di, Fan; Lingyu, Ran; Yuanzhong, Jiang; Rui, Liu; Keming, Luo

    2015-10-01

    The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.

  20. Workshop on ENU Mutagenesis: Planning for Saturation, July 25-28, 2002

    SciTech Connect

    Nadeau, Joseph H

    2002-07-25

    The goal of the conference is to enhance the development of improved technologies and new approaches to the identification of genes underlying chemically-induced mutant phenotypes. The conference brings together ENU mutagenesis experts from the United States and aborad for a small, intensive workshop to consider these issues.

  1. Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

    PubMed Central

    Valetti, Francesca; Gilardi, Gianfranco

    2013-01-01

    Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources. PMID:24970191

  2. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways

    PubMed Central

    Gómez-Marroquín, Martha; Martin, Holly A.; Pepper, Amber; Girard, Mary E.; Kidman, Amanda A.; Vallin, Carmen; Yasbin, Ronald E.; Pedraza-Reyes, Mario; Robleto, Eduardo A.

    2016-01-01

    In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu+ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu+ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome. PMID:27399782

  3. Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium

    PubMed Central

    Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž

    2016-01-01

    Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum. Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. PMID:27307293

  4. A quantitative model of bacterial mismatch repair as applied to studying induced mutagenesis

    NASA Astrophysics Data System (ADS)

    Belov, O. V.; Chuluunbaatar, O.; Kapralov, M. I.; Sweilam, N. H.

    2013-11-01

    The paper presents a mathematical model of the DNA mismatch repair system in Escherichia coli bacterial cells. The key pathways of this repair mechanism were simulated on the basis of modern experimental data. We have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Here we demonstrate an application of the model to problems of radiation-induced mutagenesis.

  5. Building on the Past, Shaping the Future: The Environmental Mutagenesis and Genomics Society

    EPA Science Inventory

    In late 2012 the members of the Environmental Mutagen Society voted to change its name to the Environmental Mutagenesis and Genomics Society. Here we describe the thought process that led to adoption of the new name, which both respects the rich history of a Society founded in 19...

  6. Identification of a halotolerant mutant via in vitro mutagenesis in the cyanobacterium Fremyella diplosiphon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. The effect of salinity on the fresh water cyanobacteria, Fremyella diplosiphon was investigated and mutagenesis-based efforts were undertaken to enhance salt toleran...

  7. CHEMICAL MUTAGENESIS AND CARCINOGENESIS: INCORPORATION OF MECHANISTIC DATA INTO RISK ASSESSMENT

    EPA Science Inventory

    CHEMICAL MUTAGENESIS AND CARCINOGENESIS: INCORPORATION OF MECHANISTIC DATA INTO RISK ASSESSMENT

    The current understanding of cancer as a genetic disease, requiring a specific set of genomic alterations for a normal cell to form a metastatic tumor, has provided the oppor...

  8. Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

    PubMed Central

    Ledermann, Raphael; Strebel, Silvan; Kampik, Clara

    2016-01-01

    ABSTRACT Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCE Mutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of

  9. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

    PubMed Central

    McAdam, R A; Weisbrod, T R; Martin, J; Scuderi, J D; Brown, A M; Cirillo, J D; Bloom, B R; Jacobs, W R

    1995-01-01

    Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen. PMID:7868221

  10. Random mutagenesis of the human immunodeficiency virus type-1 trans-activator of transcription (HIV-1 Tat).

    PubMed Central

    Siderovski, D P; Matsuyama, T; Frigerio, E; Chui, S; Min, X; Erfle, H; Sumner-Smith, M; Barnett, R W; Mak, T W

    1992-01-01

    A new method is described for the direct construction of randomly mutagenized genes by applying the polymerase chain reaction (PCR) to an oligonucleotide synthesized using doped nucleotide reservoirs. We have demonstrated the utility of this method by generating a library of mutant HIV-1 tat genes. Several arbitrarily selected, inactive tat clones were sequenced to evaluate the extent of the mutagenesis. Moreover, fourteen recombinants encoding varying levels of transcriptional trans-activator activity were isolated by transient transfection of sub-library pools into a HeLa cell line bearing an HIV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene. Sequence data revealed a spectrum of alterations including nucleotide substitutions, insertions, and deletions, suggesting that mutations arose from both the doped DNA synthesis and the subsequent PCR 'rescue' of full-length product. Sequence comparison between inactive and active Tat clones revealed a selection pressure against amino-acid substitutions within the N-terminal domains of Tat, indicating the importance of this region to trans-activation competence. In addition, single and double missense mutations within the basic-rich, TAR RNA-binding domain were seen to be tolerated within active Tat clones. Images PMID:1437550

  11. A Mutant Mouse with a Highly Specific Contextual Fear-Conditioning Deficit Found in an N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen

    ERIC Educational Resources Information Center

    Pletcher, Mathew T.; Wiltshire, Tim; Tarantino, Lisa M.; Mayford, Mark; Reijmers, Leon G.; Coats, Jennifer K.

    2006-01-01

    Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has…

  12. The dilemma of revealing sensitive information on paternity status in Arabian social and cultural contexts: telling the truth about paternity in Saudi Arabia.

    PubMed

    Adlan, Abdallah A; ten Have, Henk A M J

    2012-12-01

    Telling the truth is one of the most respected virtues in medical history and one of the most emphasized in the code of medical ethics. Health care providers are frequently confronted with the dilemma as to whether or not to tell the truth. This dilemma deepens when both choices are critically vicious: The choice is no longer between "right and right" or "right and wrong," it is between "wrong and wrong." In the case presented and discussed in this paper, a research team in Saudi Arabia unintentionally uncovered information regarding misattributed paternity. In such a situation and in the context of a tribal cultural system, what should the team do with this information? This case analysis demonstrates the joint application of ethical resources originating from within and outside the Saudi Arabian context. The article analyses the case based on the moral problems involved, relevant medical application, and the impact of such information in the Saudi tribal and Islamic domains. The most pertinent relevant values and secular debates on similar matters are discussed. Finally, the article aims to provide an Islamic dimension of family, fatherhood, and adultery.

  13. EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

    PubMed Central

    2014-01-01

    Background TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction. PMID:24475756

  14. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

    PubMed Central

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  15. Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis.

    PubMed

    Yu, Mina; Yu, Junjie; Hu, Jiankun; Huang, Lei; Wang, Yahui; Yin, Xiaole; Nie, Yafeng; Meng, Xiangkun; Wang, Weiduo; Liu, Yongfeng

    2015-03-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. To gain more insight into the cellular and molecular mechanisms in the pathogenesis of Ustilaginoidea virens, the causal agent of rice false smut disease, a T-DNA insertion mutant library of U. virens was established using ATMT. We optimized a range of conditions to improve the transformation efficiency. Transformants were most effectively obtained when the optimal co-cultivation time is 72h, with 50μM AS in medium and 100μl A. tumefaciens for co-cultivation, leading to the production of 160-185 hygromycin B resistant transformants per 1×10(5) conidia. Southern blot analysis indicated that 58.14% of transformants had a single T-DNA copy. Among 5600 transformants tested for virulence, 37 mutants with reproducible pathogenic defects were obtained. The flanking sequences of three avirulent tranformants (B20, B1015 and B1465) and two pathogenicity-reduced transformants (B726 and B785) were amplified by high-efficiency thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertion in mutant B20 targeted the coding region of a gene encoding a protein highly similar to SUN family protein, and in mutant B726 targeted upstream of a gene with unknown function. The two T-DNA insertion sites in mutant B785 were found in the coding region of a gene encoding C6 transcription factor, but failed in amplified flanking sequence of another T-DNA. Chromosomal rearrangement occurred in the genome of mutant B1016 and B1465 with single T-DNA insertion. Among avirulent mutants, B20 showed altered colony growth and pigmentation. The T-DNA insert in B20 was detected in the coding region of a gene named UvSUN2. Morphophysiological characterization analysis suggested that UvSUN2 might be a virulence factor, and possibly required for proper fungal growth, cell wall construction, and stress responses in U. virens

  16. Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

    SciTech Connect

    Michaud III, Edward J; Culiat, Cymbeline T; Klebig, Mitch; Barker, Gene; Cain, K T; Carpenter, Debra J S; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn Wallace; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert Edward; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann; Rinchik, Eugene M; Johnson, Dabney K

    2005-01-01

    Background: Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results: We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions: The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

  17. Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.

    PubMed

    Smith, Everett Clinton; Blanc, Hervé; Surdel, Matthew C; Vignuzzi, Marco; Denison, Mark R

    2013-08-01

    No therapeutics or vaccines currently exist for human coronaviruses (HCoVs). The Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) epidemic in 2002-2003, and the recent emergence of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in April 2012, emphasize the high probability of future zoonotic HCoV emergence causing severe and lethal human disease. Additionally, the resistance of SARS-CoV to ribavirin (RBV) demonstrates the need to define new targets for inhibition of CoV replication. CoVs express a 3'-to-5' exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication and is conserved across the CoV family. All genetic and biochemical data support the hypothesis that nsp14-ExoN has an RNA proofreading function. Thus, we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. We demonstrate that while wild-type (ExoN+) CoVs were resistant to RBV and 5-fluorouracil (5-FU), CoVs lacking ExoN activity (ExoN-) were up to 300-fold more sensitive. While the primary antiviral activity of RBV against CoVs was not mutagenesis, ExoN- CoVs treated with 5-FU demonstrated both enhanced sensitivity during multi-cycle replication, as well as decreased specific infectivity, consistent with 5-FU functioning as a mutagen. Comparison of full-genome next-generation sequencing of 5-FU treated SARS-CoV populations revealed a 16-fold increase in the number of mutations within the ExoN- population as compared to ExoN+. Ninety percent of these mutations represented A:G and U:C transitions, consistent with 5-FU incorporation during RNA synthesis. Together our results constitute direct evidence that CoV ExoN activity provides a critical proofreading function during virus replication. Furthermore, these studies identify ExoN as the first viral protein distinct from the RdRp that determines the sensitivity of RNA viruses to mutagens. Finally, our results show the importance of ExoN as a target for inhibition

  18. Structure-function studies on human retinol-binding protein using site-directed mutagenesis.

    PubMed Central

    Sivaprasadarao, A; Findlay, J B

    1994-01-01

    Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and

  19. Single-trial EEG-informed fMRI reveals spatial dependency of BOLD signal on early and late IC-ERP amplitudes during face recognition.

    PubMed

    Wirsich, Jonathan; Bénar, Christian; Ranjeva, Jean-Philippe; Descoins, Médéric; Soulier, Elisabeth; Le Troter, Arnaud; Confort-Gouny, Sylviane; Liégeois-Chauvel, Catherine; Guye, Maxime

    2014-10-15

    Simultaneous EEG-fMRI has opened up new avenues for improving the spatio-temporal resolution of functional brain studies. However, this method usually suffers from poor EEG quality, especially for evoked potentials (ERPs), due to specific artifacts. As such, the use of EEG-informed fMRI analysis in the context of cognitive studies has particularly focused on optimizing narrow ERP time windows of interest, which ignores the rich diverse temporal information of the EEG signal. Here, we propose to use simultaneous EEG-fMRI to investigate the neural cascade occurring during face recognition in 14 healthy volunteers by using the successive ERP peaks recorded during the cognitive part of this process. N170, N400 and P600 peaks, commonly associated with face recognition, were successfully and reproducibly identified for each trial and each subject by using a group independent component analysis (ICA). For the first time we use this group ICA to extract several independent components (IC) corresponding to the sequence of activation and used single-trial peaks as modulation parameters in a general linear model (GLM) of fMRI data. We obtained an occipital-temporal-frontal stream of BOLD signal modulation, in accordance with the three successive IC-ERPs providing an unprecedented spatio-temporal characterization of the whole cognitive process as defined by BOLD signal modulation. By using this approach, the pattern of EEG-informed BOLD modulation provided improved characterization of the network involved than the fMRI-only analysis or the source reconstruction of the three ERPs; the latter techniques showing only two regions in common localized in the occipital lobe.

  20. Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis

    PubMed Central

    Lim, Joseph B; Barker, Kimberly A; Eller, Kristen A; Jiang, Linda; Molina, Veronica; Saifee, Jessica F; Sikes, Hadley D

    2015-01-01

    As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity. PMID:26311413

  1. Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis.

    PubMed

    Lim, Joseph B; Barker, Kimberly A; Eller, Kristen A; Jiang, Linda; Molina, Veronica; Saifee, Jessica F; Sikes, Hadley D

    2015-11-01

    As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity.

  2. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  3. Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio) Reveals No Effects on Reproductive Performance

    PubMed Central

    Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

    2016-01-01

    Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form) function in zebrafish (Danio rerio) to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga) during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3-/- fish. The gnrh3-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored. PMID:27355207

  4. A strategy for fine-structure functional analysis of a 6- to 11-centimorgan region of mouse chromosome 7 by high-efficiency mutagenesis.

    PubMed

    Rinchik, E M; Carpenter, D A; Selby, P B

    1990-02-01

    A refined functional map of a 6- to 11-centimorgan region surrounding the albino (c) locus in mouse chromosome 7 is being generated by N-ethyl-N-nitrosourea (EtNU) "saturation" mutagenesis of stem-cell spermatogonia. In the first phase of an experiment that will eventually test at least 3000 gametes, we screened 972 mutagenized gametes for the induction of both lethal and visible mutations with a two-cross breeding protocol. Thirteen mutations mapping within the limits of a segment corresponding to the cytologically visible Df(c Mod-2 sh-1)26DVT deletion were recovered. They represented three phenotypic groups: prenatal lethality (six mutations); a fitness/runting syndrome (three mutations, provisionally designated as fit variants); and a neurological/balance-defect abnormality (four mutations). Complementation analysis provided evidence for a true repeat mutation at the sh-1 (shaker-1) locus (for the neurological mutations) and another at the here defined fit-1 (fitness-1) locus. In addition, four complementation groups were defined by induced lethal mutations; the two other lethal mutations were each part of a cluster. The recovery of the repeat mutations suggests that the EtNU-induced mutation rate, estimated from specific-locus tests, should make it possible to achieve saturation mutagenesis of a chromosomal region. This experiment is providing basic logistical and statistical information on which to base strategies for expanding the functional map of larger segments of the mouse genome by experimental mutagenesis. It is also yielding additional mutations useful in dissecting the functional and molecular complexity of this segment of chromosome 7.

  5. Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

    PubMed Central

    Toniti, Waraphan; Yoshida, Toru; Tsurumura, Toshiharu; Irikura, Daisuke; Monma, Chie; Kamata, Yoichi

    2017-01-01

    Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin. PMID:28199340

  6. How Are Preferences Revealed?

    PubMed Central

    Beshears, John; Choi, James J.; Laibson, David; Madrian, Brigitte C.

    2009-01-01

    Revealed preferences are tastes that rationalize an economic agent’s observed actions. Normative preferences represent the agent’s actual interests. It sometimes makes sense to assume that revealed preferences are identical to normative preferences. But there are many cases where this assumption is violated. We identify five factors that increase the likelihood of a disparity between revealed preferences and normative preferences: passive choice, complexity, limited personal experience, third-party marketing, and intertemporal choice. We then discuss six approaches that jointly contribute to the identification of normative preferences: structural estimation, active decisions, asymptotic choice, aggregated revealed preferences, reported preferences, and informed preferences. Each of these approaches uses consumer behavior to infer some property of normative preferences without equating revealed and normative preferences. We illustrate these issues with evidence from savings and investment outcomes. PMID:24761048

  7. The spatial epidemiology of trauma: the potential of geographic information science to organize data and reveal patterns of injury and services.

    PubMed

    Schuurman, Nadine; Hameed, S Morad; Fiedler, Robert; Bell, Nathaniel; Simons, Richard K

    2008-10-01

    Despite important advances in the prevention and treatment of trauma, preventable injuries continue to impact the lives of millions of people. Motor vehicle collisions and violence claim close to 3 million lives each year worldwide. Public health agencies have promoted the need for systematic and ongoing surveillance as a foundation for successful injury control. Surveillance has been used to quantify the incidence of injury for the prioritization of further research, monitor trends over time, identify new injury patterns, and plan and evaluate prevention and intervention efforts. Advances in capability to handle spatial data and substantial increases in computing power have positioned geographic information science (GIS) as a potentially important tool for health surveillance and the spatial organization of health care, and for informing prevention and acute care interventions. Two themes emerge in the trauma literature with respect to GIS theory and techniques: identifying determinants associated with the risk of trauma to guide injury prevention efforts and evaluating the spatial organization and accessibility of acute trauma care systems. We review the current literature on trauma and GIS research and provide examples of the importance of accounting for spatial scale when using spatial analysis for surveillance. The examples illustrate the effect of scale on incident analysis, the geographic variation of major injury across British Columbia's health service delivery areas (HSDAs) and the rates of variation of injury within individual HSDAs.

  8. Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway.

    PubMed

    Cooksley, Clare M; Zhang, Ying; Wang, Hengzheng; Redl, Stephanie; Winzer, Klaus; Minton, Nigel P

    2012-11-01

    The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to

  9. Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca 2+ -calmodulin

    SciTech Connect

    Lin, Jiusheng; van den Bedem, Henry; Brunger, Axel T.; Wilson, Mark A.

    2016-01-01

    Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

  10. Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza

    PubMed Central

    Li, Bin; Cui, Guanghong; Shen, Guoan; Zhan, Zhilai; Huang, Luqi; Chen, Jiachun; Qi, Xiaoquan

    2017-01-01

    CRISPR/Cas9 is a powerful genome editing tool that has been extensively used in model plants and crops, such as Arabidopsis thaliana, rice, wheat, and soybean. Here, we report the use of CRISPR/Cas9 to precisely knock out the committed diterpene synthase gene (SmCPS1) involved in tanshinone biosynthesis in Salvia miltiorrhiza, a traditional Chinese medicinal herb with significant pharmacological activities, such as vasorelaxation, protection against ischemia-reperfusion injury, and antiarrhythmic effects. Three homozygous and eight chimeric mutants were obtained from 26 independent transgenic hairy root lines by Agrobacterium rhizogenes-mediated transformation. The metabolomic analysis based on LC-qTOF-MS and Q-TRAP-LC-MS/MS revealed that tanshinones, especially cryptotanshinone, tanshinone IIA and tanshinone I, are completely missing in homozygous mutants, without influencing other phenolic acid metabolites. By contrast, tanshinones are decreased but still detectable in chimeric mutants, which is similar to a previously-reported an RNAi study of SmCPS1. These results demonstrate that Agrobacterium rhizogenes- mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in S. miltiorrhiza, thus paving the way for large-scale genome editing in S. miltiorrhiza, which is important for pathway elucidation of secondary metabolites, quality improvement, and yield increases for this valuable traditional Chinese medicinal herb. PMID:28256553

  11. Mutual information analysis reveals bigeminy patterns in Andersen-Tawil syndrome and in subjects with a history of sudden cardiac death

    NASA Astrophysics Data System (ADS)

    Núñez-Acosta, Elisa; Lerma, Claudia; Márquez, Manlio F.; José, Marco V.

    2012-02-01

    Herein we introduce the Mutual Information Function (MIF) as a mathematical method to analyze ventricular bigeminy in certain pathological conditions of the heart known to be associated with frequent ventricular arrhythmias. In particular, we show that the MIF is sensitive enough to detect the bigeminy pattern in symbolic series from patients with Andersen-Tawil syndrome as well as in a group of patients from the Sudden Cardiac Death Holter Databases. The results confirm that MIF is an adequate method to detect the autocorrelation between the appearance of sinus and ventricular premature beats resulting in a bigeminy pattern. It is also shown that MIF reflects the bigeminy patterns as a function of the percentage of ventricular premature beats present in the symbolic series and also as a function of the percentage of bigeminy. The MIF was also useful to establish a consistent difference in the bigeminy pattern related to the diurnal and nocturnal periods presumably associated to the circadian rhythm of the heart. Understanding of the ventricular bigeminy patterns throughout 24-hours could provide some insights into the pathogenesis of ventricular tachyarrhythmias in these pathological conditions.

  12. Ultraviolet Mutagenesis in Bacteriophage T4 I. Irradiation of Extracellular Phage Particles

    PubMed Central

    Drake, John W.

    1966-01-01

    Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. I. Irradiation of extracellular phage particles. J. Bacteriol. 91:1775–1780. 1966.—Ultraviolet (UV) irradiation of extracellular T4 phage particles induces about 2 × 10−4r mutations per lethal hit. The mutants largely escape detection unless the irradiated phages are plated with very soft overlay agar. Multiplicity reactivation is not a prerequisite for mutagenesis. A much higher frequency of base pair substitution-type mutants is induced than is found in the spontaneous background, but sign mutants are also induced. Nearly half of the mutants map into previously identified UV hot spots. The rII mutants induced extracellularly are very similar to those induced intracellularly. The mutants also appear to result from direct radiation effects upon the bacteriophage deoxyribonucleic acid. PMID:5937237

  13. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences.

  14. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis

    PubMed Central

    Beacham, T.A.; Macia, V. Mora; Rooks, P.; White, D.A.; Ali, S.T.

    2015-01-01

    Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed. PMID:26753128

  15. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    PubMed Central

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation. PMID:11172002

  16. Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

    PubMed Central

    Balhana, Ricardo; Stoker, Neil G.; Sikder, Mahmudul Hasan; Chauviac, Francois-Xavier; Kendall, Sharon L.

    2010-01-01

    Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis. PMID:20650290

  17. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.

  18. Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression

    PubMed Central

    Rangel, Roberto; Lee, Song-Choon; Hon-Kim Ban, Kenneth; Guzman-Rojas, Liliana; Mann, Michael B.; Newberg, Justin Y.; McNoe, Leslie A.; Selvanesan, Luxmanan; Ward, Jerrold M.; Rust, Alistair G.; Chin, Kuan-Yew; Black, Michael A.; Jenkins, Nancy A.; Copeland, Neal G.

    2016-01-01

    Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC. PMID:27849608

  19. Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis.

    PubMed

    Erlacher, Matthias D; Chirkova, Anna; Voegele, Paul; Polacek, Norbert

    2011-05-01

    The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

  20. [RAD18 gene product of yeast Saccharomyces cerevisiae controls mutagenesis induced by hydrogen peroxide].

    PubMed

    Kozhina, T N; Korolev, V G

    2012-04-01

    Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6-Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.

  1. Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.

    PubMed

    Kille, Sabrina; Acevedo-Rocha, Carlos G; Parra, Loreto P; Zhang, Zhi-Gang; Opperman, Diederik J; Reetz, Manfred T; Acevedo, Juan Pablo

    2013-02-15

    Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

  2. Novobiocin Inhibits the Antimicrobial Resistance Acquired through DNA Damage-Induced Mutagenesis in Acinetobacter baumannii

    PubMed Central

    Jara, Luis M.; Pérez-Varela, María; Corral, Jordi; Arch, Marta; Cortés, Pilar; Bou, Germán; Barbé, Jordi

    2015-01-01

    Acinetobacter baumannii, a worldwide emerging nosocomial pathogen, acquires antimicrobial resistances in response to DNA-damaging agents, which increase the expression of multiple error-prone DNA polymerase components. Here we show that the aminocoumarin novobiocin, which inhibits the DNA damage response in Gram-positive bacteria, also inhibits the expression of error-prone DNA polymerases in this Gram-negative multidrug-resistant pathogen and, consequently, its potential acquisition of antimicrobial resistance through DNA damage-induced mutagenesis. PMID:26503651

  3. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways. PMID:26733980

  4. [Cloning and insertion mutagenesis of DNA fragment coding for the luminescent system of Photobacterium leiognathi].

    PubMed

    Ptitsyn, L R; Gurevich, V B; Barsanova, T G; Shenderov, A N; Khaĭkinson, M Ia

    1988-10-01

    Fragments of DNA, obtained from the luminescent bacterium Photobacterium leiognathi and inserted into the plasmid pBR322, were found to code for the luminescence expressed in E. coli cells. The genetic functions necessary for light production in E. coli are localized on a DNA fragment of about 7 kbp. The insertion mutagenesis was used to define the luminescence functions encoded by the hybrid plasmid.

  5. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

    PubMed

    Monson, Rita; Smith, Debra S; Matilla, Miguel A; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P C

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

  6. Steady-state transposon mutagenesis in inbred maize.

    PubMed

    McCarty, Donald R; Settles, Andrew Mark; Suzuki, Masaharu; Tan, Bao Cai; Latshaw, Susan; Porch, Tim; Robin, Kevin; Baier, John; Avigne, Wayne; Lai, Jinsheng; Messing, Joachim; Koch, Karen E; Hannah, L Curtis

    2005-10-01

    We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.

  7. X-Ray Structure and Site-Directed Mutagenesis Analysis of the Escherichia coli Colicin M Immunity Protein▿ †

    PubMed Central

    Gérard, Fabien; Brooks, Mark A.; Barreteau, Hélène; Touzé, Thierry; Graille, Marc; Bouhss, Ahmed; Blanot, Didier; van Tilbeurgh, Herman; Mengin-Lecreulx, Dominique

    2011-01-01

    Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four β strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated. PMID:21037007

  8. Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis

    PubMed Central

    Ipsaro, Jonathan J.; Shen, Chen; Arai, Eri; Xu, Yali; Kinney, Justin B.; Joshua-Tor, Leemor; Vakoc, Christopher R.

    2017-01-01

    Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development. PMID:28231254

  9. Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis.

    PubMed

    Ipsaro, Jonathan J; Shen, Chen; Arai, Eri; Xu, Yali; Kinney, Justin B; Joshua-Tor, Leemor; Vakoc, Christopher R; Shi, Junwei

    2017-01-01

    Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.

  10. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

    PubMed

    Sanchis, Joaquin; Fernández, Layla; Carballeira, J Daniel; Drone, Jullien; Gumulya, Yosephine; Höbenreich, Horst; Kahakeaw, Daniel; Kille, Sabrina; Lohmer, Renate; Peyralans, Jérôme J-P; Podtetenieff, John; Prasad, Shreenath; Soni, Pankaj; Taglieber, Andreas; Wu, Sheng; Zilly, Felipe E; Reetz, Manfred T

    2008-11-01

    Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

  11. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    PubMed

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement.

  12. Inhibitory effects of chlorophyllin on 7,12-dimethylbenz[a]anthracene-induced bacterial mutagenesis and mouse skin carcinogenesis.

    PubMed

    Chung, W Y; Lee, J M; Park, M Y; Yook, J I; Kim, J; Chung, A S; Surh, Y J; Park, K K

    1999-10-18

    Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been used for the treatment of several abnormal human conditions without apparent toxicity. Recent studies have revealed that CHL has the excellent chemopreventive potential. In the present investigation, we have found the inhibitory activities of CHL against 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in Salmonella typhimurium TA100 and also on DMBA-initiated and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-promoted mouse skin tumor formation. The incidence and the multiplicity of skin tumors were not significantly decreased in mice by a single topical application of CHL prior to the DMBA treatment, but there was a marked suppression of papillomagenesis in mice treated with CHL during the promotional stage. Furthermore, the formation of DMBA-induced papillomagenesis was reduced in all mice that had received CHL for 6 weeks following treatment with TPA for 6, 18 and 24 weeks. These results indicate that CHL can inhibit both tumor promotion and the progression of papillomagenesis in the two-stage mouse skin carcinogenesis induced by DMBA and TPA.

  13. The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes.

    PubMed

    Hardré, Hélène; Kuhn, Lauriane; Albrieux, Catherine; Jouhet, Juliette; Michaud, Morgane; Seigneurin-Berny, Daphné; Falconet, Denis; Block, Maryse A; Maréchal, Eric

    2014-01-01

    The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM-OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.

  14. The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes

    PubMed Central

    Hardré, Hélène; Kuhn, Lauriane; Albrieux, Catherine; Jouhet, Juliette; Michaud, Morgane; Seigneurin-Berny, Daphné; Falconet, Denis; Block, Maryse A.; Maréchal, Eric

    2014-01-01

    The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM–OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM–OEM complexes in various physiological contexts and using virtually any other purified membrane organelle. PMID:24999344

  15. Pseudorabies virus glycoprotein C attachment-proficient revertants isolated through a simple, targeted mutagenesis scheme.

    PubMed

    Rue, Cary A; Ryan, Patrick

    2008-07-01

    Pseudorabies virus (PRV) glycoprotein C (gC) initiates virus attachment to cells by binding to heparan sulfate (HS) proteoglycans. The gC:HS interaction is not essential since gC null mutants still infect; however, they are more easily removed from cells during the initial stages of infection. The expendability of gC has facilitated a genetic mapping of the HS-binding domain, which is composed of three independent heparin-binding domains (HBDs) of six to eight amino acids each. Previous results suggested that at least one of the HBDs (HBD 1) functioned in a context-dependent manner. To define the context better, a reversion analysis was performed in which a defective gC containing a nonfunctional but intact HBD 1 regained HS-binding ability. To increase the reversion frequency, an efficient method for targeted, yet random mutagenesis of the gC gene was developed. The method involves random mutagenesis of a plasmid-borne copy of gC, and highly efficient recombination of the plasmid-borne genes into the virus genome at the site of a double-strand break in the viral gC locus. Revertants were recovered readily, and their gC alleles suggested that HS-binding could be restored by several different amino acid substitutions. This approach should be applicable to targeted mutagenesis of other herpesvirus genes.

  16. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    PubMed

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-06

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato.

  17. Ascorbate enhances u. v. -mutagenesis in E. coli but inhibits it in Chinese hamster cells

    SciTech Connect

    Rossman, T.G.; Klein, C.B.; Naslund, M.

    1986-05-01

    Ascorbic acid (vitamin C) causes an increase in the mutation frequency of u.v.-irradiated Escherichia coli WP2. The enhancement occurs at all u.v. fluences, and is dependent upon the ascorbate concentration in the medium. A maximum effect (approximately 8- to 13-fold) is seen at 100-150 micrograms/ml, although some enhancement can be seen even at 10 micrograms/ml. The comutagenic effect of ascorbate with u.v. in E. coli is dependent upon peptone, a constituent of nutrient broth. The enhancement of u.v.-mutagenesis by ascorbate is absent in strains WP2s (uvrA) and WP6 (polA), suggesting that ascorbate affects the repair of pyrimidine dimers. The opposite results are observed for u.v.-mutagenesis in Chinese hamster V79 cells. The presence of ascorbate (50 micrograms/ml) during u.v. irradiation does not enhance the u.v. effect, but rather decreases it approximately 30%. These results are discussed with regard to differences in the mechanism of u.v.-mutagenesis and DNA repair in bacterial and mammalian cells.

  18. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

    PubMed Central

    Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

    2017-01-01

    The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

  19. Dissecting partner recognition by an intrinsically disordered protein using descriptive random mutagenesis.

    PubMed

    Gruet, Antoine; Dosnon, Marion; Vassena, Andrea; Lombard, Vincent; Gerlier, Denis; Bignon, Christophe; Longhi, Sonia

    2013-09-23

    In view of getting insights into the molecular determinants of the binding efficiency of intrinsically disordered proteins (IDPs), we used random mutagenesis. As a proof of concept, we chose the interaction between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein and assessed how amino acid substitutions introduced at random within NTAIL affect partner recognition. In contrast with directed evolution approaches, we did not apply any selection and used the gene library approach not for production purposes but for achieving a better understanding of the NTAIL/XD interaction. For that reason, and to differentiate our approach from similar approaches that make use of systematic (i.e., targeted) mutagenesis, we propose to call it "descriptive random mutagenesis" (DRM). NTAIL variants generated by error-prone PCR were picked at random in the absence of selection pressure and were characterized in terms of sequence and binding abilities toward XD. DRM not only identified determinants of NTAIL/XD interaction that were in good agreement with previous work but also provided new insights. In particular, we discovered that the primary interaction site is poorly evolvable in terms of binding abilities toward XD. We also identified a critical NTAIL residue whose role in stabilizing the NTAIL/XD complex had previously escaped detection, and we identified NTAIL regulatory sites that dampen the interaction while being located outside the primary interaction site. Results show that DRM is a valuable approach to study binding abilities of IDPs.

  20. 5-fluorouracil in lethal mutagenesis of foot-and-mouth disease virus.

    PubMed

    Agudo, Rubén; Arias, Armando; Domingo, Esteban

    2009-06-01

    5-fluorouracil (FU) is a pyrimidine analogue extensively used in cancer chemotherapy. FU can be metabolized into 5-fluorouridine-triphosphate, which can be used as substrate for viral RNA-dependent RNA polymerases. This results in the incorporation of mutations into viral RNA. Accumulation of mutations may lead to loss of virus infectivity, in a process known as lethal mutagenesis. RNA virus pathogens are particularly difficult to control because they are highly mutable, and mutants resistant to antiviral agents are readily selected. Here, we review the basic principles of lethal mutagenesis as an antiviral approach, and the participation of FU in its development. Recent studies with foot-and-mouth disease virus indicate that FU can act both as an inhibitor and as a mutagen during foot-and-mouth disease virus replication. This dual activity renders FU an adequate drug for lethal mutagenesis. We suggest that structural and biochemical studies can contribute to the lead to new design of base or nucleoside analogues targeted specifically to viral polymerases.

  1. Saturation mutagenesis of selected residues of the α-peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity

    PubMed Central

    Field, Des; Molloy, Evelyn M; Iancu, Catalin; Draper, Lorraine A; O' Connor, Paula M; Cotter, Paul D; Hill, Colin; Ross, R Paul

    2013-01-01

    Summary The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post-translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram-positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering. Funding Information Work in the authors' laboratory is supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H and R.P.R. with SFI Principal Investigator funding. PMID:23433070

  2. Mutagenesis by outer space parameters other than cosmic rays

    NASA Astrophysics Data System (ADS)

    Horneck, Gerda; Rabbow, Elke

    We have studied the ability of microorganisms to cope with the complex interplay of the parameters of space in experiments in low Earth orbit and using space simulation facilities on ground. Emphasis was laid on space parameters other than cosmic rays. The studies are directed towards understanding prebiotic chemical evolution and biological evolution processes, and interplanetary transfer of life. Effects of space vacuum: Space experiments have shown that up to 70% of bacterial and fungal spores survived short-term exposure to space vacuum. The chances of survival in space were increased when spores were embedded in chemical protectants such as sugars, or salt crystals, or when they were exposed in multilayer. During the six years lasting LDEF mission up to 80% of bacterial spores survived exposure to space vacuum. A 10-fold increased mutation rate over the spontaneous rate has been observed in spores of Bacillus subtilis after exposure to space vacuum, which is probably based on a unique molecular signature of tandem-double base change at restricted sites in the DNA. In addition, DNA strand breaks have been observed to be induced by vacuum treatment. Effects of extraterrestrial solar UV radiation: Solar UV radiation has been found to be the most deleterious factor of space. The reason for this is the highly energetic UV-C and vacuum UV radiation that is directly absorbed by the DNA and which induces specific photoproducts in the DNA that are highly mutagenic and lethal. The damaging effect of extraterrestrial solar UV radiation was even aggravated, when the spores were simultaneously exposed to both, solar UV radiation and space vacuum. In order to investigate the mutagenic potential of solar UV radiation, DNA of the Escherichia coli plasmid pUC19 was exposed to selected wavebands of UV radiation (from vacuum UV to UV-A) by use of a solar simulator and space simulation facilities. Action spectra revealed that for vacuum UV different kinds of photochemical damage

  3. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

  4. Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka.

    PubMed

    Ansai, Satoshi; Inohaya, Keiji; Yoshiura, Yasutoshi; Schartl, Manfred; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2014-01-01

    Genome editing using engineered nucleases such as transcription activator-like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.

  5. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

    PubMed Central

    Boretsky, Yuriy R.; Pynyaha, Yuriy V.; Boretsky, Volodymyr Y.; Fedorovych, Dariya V.; Fayura, Lyubov R.; Protchenko, Olha; Philpott, Caroline C.; Sibirny, Andriy A.

    2012-01-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Δvma1–17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Δfra1–45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1–17 and Δfes1–77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1–45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1–77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80–22. Complementation analysis revealed that Δvma1–17 and Δfra1–45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  6. Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

    PubMed Central

    Sainz, M B; Goff, S A; Chandler, V L

    1997-01-01

    C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains. PMID:8972191

  7. Gene-trap mutagenesis using Mol/MSM-1 embryonic stem cells from MSM/Ms mice.

    PubMed

    Nakahara, Mai; Tateyama, Hiroki; Araki, Masatake; Nakagata, Naomi; Yamamura, Ken-ichi; Araki, Kimi

    2013-06-01

    The MSM/Ms strain is derived from the Japanese wild mouse Mus musculus molossinus and displays characteristics not observed in common laboratory strains. Functional genomic analyses using genetically engineered MSM/Ms mice will reveal novel phenotypes and gene functions/interactions. We previously reported the establishment of a germline-competent embryonic stem (ES) cell line, Mol/MSM-1, from the MSM/Ms strain. To analyze its usefulness for insertional mutagenesis, we performed gene-trapping using these cells. In the present study, we compared the gene-trap events between Mol/MSM-1 and a conventional ES cell line, KTPU8, derived from the F1 progeny of a C57BL/6 × CBA cross. We introduced a promoter-trap vector carrying the promoterless β-galactosidase/neomycin-resistance fusion gene into Mol/MSM-1 and KTPU8 cells, isolated clones, and identified the trapped genes by rapid amplification of cDNA 5'-ends (5'-RACE), inverse PCR, or plasmid rescue. Unexpectedly, the success rate of 5'-RACE in Mol/MSM trap clones was 47 %, lower than the 87 % observed in KTPU8 clones. Genomic analysis of the 5'-RACE-failed clones revealed that most had trapped ribosomal RNA gene regions. The percentage of ribosomal RNA region trap clones was 41 % in Mol/MSM-1 cells, but less than 10 % in KTPU8 cells. However, within the Mol/MSM-1 5'-RACE-successful clones, the trapping frequency of annotated genes, the chromosomal distribution of vector insertions, the frequency of integration into an intron around the start codon-containing exon, and the functional spectrum of trapped genes were comparable to those in KTPU8 cells. By selecting 5'-RACE-successful clones, it is possible to perform gene-trapping efficiently using Mol/MSM-1 ES cells and promoter-trap vectors.

  8. Reciprocal relationship between mouse germ-cell mutagenesis and basic genetics: from early beginnings to future opportunities.

    PubMed

    Russell, L B

    1989-01-01

    The scientific foundations for several mammalian germ-line mutagenesis tests in common use today were laid in the 1930s, 1940s, and early 1950s. Subsequent developments in the field have had multiple objectives: detection of mutagenicity of environmental agents (which has led to the development of numerous methodologies), identification of biological and physical factors that affect mutation yield, analysis of the structural nature of the genetic alterations, and assessment of the organismic effects of various types of mutations. Mutagenesis studies have made numerous contributions to basic genetics by generating mutant types that led to elucidation of sex-determining mechanisms in mammals; formulation of the single-active-, or inactive-, X-chromosome hypothesis; correlation of genetic and cytological maps; discovery of genetic "imprinting" phenomena; study of developmental pathways and cell lineages, etc. Particularly useful are sets of complexly overlapping deletions that have been recovered in radiation mutagenesis studies, propagated in breeding stocks, and genetically analyzed; these have constituted prerequisites for molecular genetic studies aimed at development of the DNA structure-function relationships for important genomic regions. Mutagenesis experiments have also served to identify mutagens that are particularly effective in inducing specific types of genetic lesions desired for basic studies. Reciprocally, basic genetics has contributed to the development of mutagenesis tests and has enhanced the value of the specific-locus test by adding to its quantitative capabilities the capability for qualitatively characterizing the actions of mutagens.

  9. Enhancing effect of heterocyclic amines and beta-carbolines on UV or chemically induced mutagenesis in E. coli.

    PubMed

    Shimoi, K; Kawabata, H; Tomita, I

    1992-08-01

    Most heterocyclic amines and beta-carbolines--harman, norharman, harmine, harmaline--enhanced UVC (254 nm) induced mutagenesis without microsomal activation in E. coli B/r WP2. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was most effective and increased UVAB (295-400 nm) induced mutations as well as UVC induced ones. Trp-P-1 enhanced the frequencies of mutations induced by not only UV but also 4-nitroquinoline-1-oxide (4NQO) or 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF2), while it showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or gamma-ray induced mutagenesis. Trp-P-1 decreased the survival of UVC irradiated cells of CM571recA. However, these effects of Trp-P-1 on UVC induced mutagenesis and lethality were not observed in WP2suvrA which is excision repair deficient. The alkaline sucrose gradient sedimentation analysis demonstrated that Trp-P-1 blocked the incision step in DNA excision repair. Further, pretreatment with Trp-P-1 before UVC irradiation showed no effect on UVC induced mutagenesis. Similar effects were also seen in the case of harman or norharman. These results suggest that heterocyclic amines and beta-carbolines inhibit DNA excision repair directly or indirectly, thus enhancing UV or chemically induced mutagenesis.

  10. Kinetic and site-directed mutagenesis studies of the cysteine residues of bovine low molecular weight phosphotyrosyl protein phosphatase.

    PubMed

    Davis, J P; Zhou, M M; Van Etten, R L

    1994-03-25

    The roles of the 8 conserved cysteines and 1 arginine in the low molecular weight phosphotyrosyl protein phosphatases were investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for Cys-12, Cys-17, and Arg-18. The CD spectra of the purified proteins were effectively superimposable, consistent with the conclusion that no major structural alterations had occurred, but 1H NMR spectroscopy did reveal some spectral shifts in the aromatic region. Kinetic analysis of the mutant proteins demonstrated that only Cys-12, Cys-17, and Arg-18 had significantly altered catalytic activity toward the substrate p-nitrophenyl phosphate at pH 5. The Cys-12 and Arg-18 mutants were effectively inactive. Thus, it is concluded that Cys-12 is the catalytic nucleophile, and Arg-18 presumably serves an essential function in substrate binding. The C17S mutant had 6% residual activity compared with wild type protein, whereas the C17A mutant had 37% activity. Consistent with the observed activity of the Cys-17 mutant, a covalent phosphocysteine intermediate was trapped and identified by 31P NMR. Further kinetic analysis of C17A using several aryl phosphate monoester substrates with different leaving group pK alpha values indicated that no change in the rate-determining step of the catalytic mechanism had occurred, that is, dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. The C17A mutant had a 4-fold higher phosphate Ki and slightly higher Km values for p-nitrophenyl phosphate suggesting that Cys-17 may be important for optimal positioning of the substrate phosphate moiety.

  11. Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis.

    PubMed

    Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

    2007-07-01

    Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).

  12. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

    PubMed Central

    Mohr, C D; Deretic, V

    1990-01-01

    A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source. Images PMID:2121708

  13. Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis*

    PubMed Central

    Ogren, John I.; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L.; Rothschild, Kenneth J.

    2015-01-01

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2380 state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2380 formation. The unusual charge neutrality of both Schiff base counterions in the P2380 conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  14. Biological effects of naturally occurring and man-made fibres: in vitro cytotoxicity and mutagenesis in mammalian cells

    PubMed Central

    Okayasu, R; Wu, L; Hei, T K

    1999-01-01

    Cytotoxicity and mutagenicity of tremolite, erionite and the man-made ceramic (RCF-1) fibre were studied using the human– hamster hybrid A L cells. Results from these fibres were compared with those of UICC Rhodesian chrysotile fibres. The A L cell mutation assay, based on the S1 gene marker located on human chromosome 11, the only human chromosome contained in the hybrid cell, has been shown to be more sensitive than conventional assays in detecting deletion mutations. Tremolite, erionite and RCF-1 fibres were significantly less cytotoxic to A L cells than chrysotile. Mutagenesis studies at the HPRT locus revealed no significant mutant yield with any of these fibres. In contrast, both erionite and tremolite induced dose-dependent S1− mutations in fibre-exposed cells, with the former inducing a significantly higher mutant yield than the latter fibre type. On the other hand, RCF-1 fibres were largely non-mutagenic. At equitoxic doses (cell survival at ∼ 0.7), erionite was found to be the most potent mutagen among the three fibres tested and at a level comparable to that of chrysotile fibres. These results indicate that RCF-1 fibres are non-genotoxic under the conditions used in the studies and suggest that the high mesothelioma incidence previously observed in hamster may either be a result of selective sensitivity of hamster pleura to fibre-induced chronic irritation or as a result of prolonged fibre treatment. Furthermore, the relatively high mutagenic potential for erionite is consistent with its documented carcinogenicity. © 1999 Cancer Research Campaign PMID:10188871

  15. Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

    PubMed

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2015-05-15

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

  16. Phenotypic Mutants of the Intracellular Actinomycete Rhodococcus equi Created by In Vivo Himar1 Transposon Mutagenesis

    PubMed Central

    Ashour, Joseph; Hondalus, Mary K.

    2003-01-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential. PMID:12670990

  17. Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli.

    PubMed

    Pai, Jen C; Entzminger, Kevin C; Maynard, Jennifer A

    2012-02-15

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3' primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.

  18. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida

    PubMed Central

    Endo, Akira; Masafumi, Mikami; Kaya, Hidetaka; Toki, Seiichi

    2016-01-01

    CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants. PMID:27905529

  19. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery.

    PubMed

    Moriarity, Branden S; Largaespada, David A

    2015-02-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS.

  20. The disparity mutagenesis model predicts rescue of living things from catastrophic errors

    PubMed Central

    Furusawa, Mitsuru

    2014-01-01

    In animals including humans, mutation rates per generation exceed a perceived threshold, and excess mutations increase genetic load. Despite this, animals have survived without extinction. This is a perplexing problem for animal and human genetics, arising at the end of the last century, and to date still does not have a fully satisfactory explanation. Shortly after we proposed the disparity theory of evolution in 1992, the disparity mutagenesis model was proposed, which forms the basis for an explanation for an acceleration of evolution and species survival. This model predicts a significant increase of the mutation threshold values if the fidelity difference in replication between the lagging and leading strands is high enough. When applied to biological evolution, the model predicts that living things, including humans, might overcome the lethal effect of accumulated deleterious mutations and be able to survive. Artificially derived mutator strains of microorganisms, in which an enhanced lagging-strand-biased mutagenesis was introduced, showed unexpectedly high adaptability to severe environments. The implications of the striking behaviors shown by these disparity mutators will be discussed in relation to how living things with high mutation rates can avoid the self-defeating risk of excess mutations. PMID:25538731

  1. Mutagenesis by host antimicrobial peptides: insights into microbial evolution during chronic infections

    PubMed Central

    Limoli, Dominique H.; Wozniak, Daniel J.

    2014-01-01

    Antimicrobial peptides (AMPs) are produced by the mammalian immune system to fight invading pathogens. The best understood function of AMPs is to integrate into the membranes of microbes, thereby disrupting and killing cells. However, a recent study [PLoS Pathogens (2014) 10, e1004083] provides evidence that at subinhibitory levels, AMPs promote mutations in bacterial DNA, which enhance bacterial survival. In particular, in the bacterium Pseudomonas aeruginosa, one AMP called LL-37 can promote mutations, which enable the bacteria to overproduce a protective sugar coating, a process called mucoid conversion. P. aeruginosa mucoid conversion is a major risk factor for those suffering from cystic fibrosis (CF), one of the most common lethal, heritable diseases in the US. LL-37 was found to produce mutations by penetrating the bacterial cell and binding to bacterial DNA. It was proposed that LL-37 binding DNA disrupts normal DNA replication and potentiates mutations. Importantly, LL-37 induced mutagenesis was also found to promote resistance to rifampicin in both P. aeruginosa and E. coli. This suggests that AMP-induced mutagenesis may be important for a broad range of chronic diseases and pathogens. PMID:28357249

  2. Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

    PubMed

    Nomura, Toshihisa; Sakurai, Tetsuya; Osakabe, Yuriko; Osakabe, Keishi; Sakakibara, Hitoshi

    2016-12-01

    Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.

  3. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

  4. Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

    PubMed

    Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

    2016-01-01

    We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

  5. Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; DeMarini, D. M.

    1999-01-01

    Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

  6. Molecular dynamics simulation and site-directed mutagenesis of alcohol acyltransferase: a proposed mechanism of catalysis.

    PubMed

    Morales-Quintana, Luis; Nuñez-Tobar, María Ximena; Moya-León, María Alejandra; Herrera, Raúl

    2013-10-28

    Aroma in Vasconcellea pubescens fruit is determined by esters, which are the products of catalysis by alcohol acyltransferase (VpAAT1). VpAAT1 protein structure displayed the conserved HxxxD motif facing the solvent channel in the center of the structure. To gain insight into the role of these catalytic residues, kinetic and site-directed mutagenesis studies were carried out in VpAAT1 protein. Based on dead-end inhibition studies, the kinetic could be described in terms of a ternary complex mechanism with the H166 residue as the catalytic base. Kinetic results showed the lowest Km value for hexanoyl-CoA. Additionally, the most favorable predicted substrate orientation was observed for hexanoyl-CoA, showing a coincidence between kinetic studies and molecular docking analysis. Substitutions H166A, D170A, D170N, and D170E were evaluated in silico. The solvent channel in all mutant structures was lost, showing large differences with the native structure. Molecular docking and molecular dynamics simulations were able to describe unfavored energies for the interaction of the mutant proteins with different alcohols and acyl-CoAs. Additionally, in vitro site-directed mutagenesis of H166 and D170 in VpAAT1 induced a loss of activity, confirming the functional role of both residues for the activity, H166 being directly involved in catalysis.

  7. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

    PubMed Central

    2013-01-01

    Background We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). Results We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Conclusions Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines. PMID:24025402

  8. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  9. Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

    PubMed Central

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2016-01-01

    Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  10. Mutagenesis and functional selection protocols for directed evolution of proteins in E. coli.

    PubMed

    Troll, Chris; Alexander, David; Allen, Jennifer; Marquette, Jacob; Camps, Manel

    2011-03-16

    The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>10(11)) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

  11. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

    PubMed Central

    Dang, Jinjun; Wei, Lei; de Ridder, Jeroen; Su, Xiaoping; Rust, Alistair G.; Roberts, Kathryn G.; Payne-Turner, Debbie; Cheng, Jinjun; Ma, Jing; Qu, Chunxu; Wu, Gang; Song, Guangchun; Huether, Robert G.; Schulman, Brenda; Janke, Laura; Zhang, Jinghui; Downing, James R.; van der Weyden, Louise; Adams, David J.

    2015-01-01

    Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL. PMID:25855603

  12. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    PubMed

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

  13. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

    PubMed Central

    Moriarity, Branden S; Largaespada, David A

    2016-01-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. PMID:26051241

  14. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    PubMed

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

  15. A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica.

    PubMed

    Neuvéglise, C; Nicauda, J M; Ross-Macdonald, P; Gaillardin, C

    1998-06-15

    A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.

  16. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

    PubMed Central

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F. Peter; Zhang, Huidong

    2017-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

  17. Control of mammalian cell mutagenesis and differentiation by chemicals which initiate or promote tumor formation

    SciTech Connect

    Jones, C. A.; Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, Chinese hamster V79 cells, which are susceptible to mutagenesis, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons and nitrosamines and to study the organ specificity exhibited by some chemical carcinogens. However, most short-term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  18. MouseNet database: digital management of a large-scale mutagenesis project.

    PubMed

    Pargent, W; Heffner, S; Schäble, K F; Soewarto, D; Fuchs, H; Hrabé de Angelis, M

    2000-07-01

    The Munich ENU Mouse Mutagenesis Screen is a large-scale mutant production, phenotyping, and mapping project. It encompasses two animal breeding facilities and a number of screening groups located in the general area of Munich. A central database is required to manage and process the immense amount of data generated by the mutagenesis project. This database, which we named MouseNet(c), runs on a Sybase platform and will finally store and process all data from the entire project. In addition, the system comprises a portfolio of functions needed to support the workflow management of the core facility and the screening groups. MouseNet(c) will make all of the data available to the participating screening groups, and later to the international scientific community. MouseNet(c) will consist of three major software components:* Animal Management System (AMS)* Sample Tracking System (STS)* Result Documentation System (RDS)MouseNet(c) provides the following major advantages:* being accessible from different client platforms via the Internet* being a full-featured multi-user system (including access restriction and data locking mechanisms)* relying on a professional RDBMS (relational database management system) which runs on a UNIX server platform* supplying workflow functions and a variety of plausibility checks.

  19. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis.

  20. Mutagenesis breeding of high echinocandin B producing strain and further titer improvement with culture medium optimization.

    PubMed

    Zou, Shu-Ping; Zhong, Wei; Xia, Chao-Jie; Gu, Ya-Nan; Niu, Kun; Zheng, Yu-Guo; Shen, Yin-Chu

    2015-10-01

    A combination of microbial strain improvement and statistical optimization is investigated to maximize echinocandin B (ECB) production from Aspergillus nidulans ZJB-0817. A classical sequential mutagenesis was studied first by using physical (ultraviolet irradiation at 254 nm) and chemical mutagens (lithium chloride and sodium nitrite). Mutant strain ULN-59 exhibited 2.1-fold increase in ECB production to 1583.1 ± 40.9 mg/L when compared with the parent strain (750.8 ± 32.0 mg/L). This is the first report where mutagenesis is applied in Aspergillus to improve ECB production. Further, fractional factorial design and central composite design were adopted to optimize the culture medium for increasing ECB production by the mutant ULN-59. Results indicated that four culture media including peptone, K2HPO4, mannitol and L-ornithine had significant effects on ECB production. The optimized medium provided another 1.4-fold increase in final ECB concentration to 2285.6 ± 35.6 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively ECB production from A. nidulans and could be a promising tool to improve other secondary metabolites production by fungal strains.

  1. Transposon-directed base-exchange mutagenesis (TDEM): a novel method for multiple-nucleotide substitutions within a target gene.

    PubMed

    Kim, Yun Cheol; Lee, Hui Sun; Yoon, Sukjoon; Morrison, Sherie L

    2009-06-01

    In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout the gene. Additionally, we developed a simple frame-checking procedure that eliminates nonfunctional mutants containing frameshifts or stop codons. As a proof of principle, we used TDEM to generate mutant lacZalpha lacking alpha-complementation activity and recovered active revertants using a second round of TDEM. Furthermore, a single round of TDEM yielded unique, inactive mutants of ccdB.

  2. TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol.

    PubMed

    Jung, Je Hyeong; Altpeter, Fredy

    2016-09-01

    Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production. Reduction of lignin content significantly improves the conversion of lignocellulosic biomass into ethanol. Conventional mutagenesis is not expected to confer reduction in lignin content in sugarcane due to its high polyploidy (x = 10-13) and functional redundancy among homo(eo)logs. Here we deploy transcription activator-like effector nuclease (TALEN) to induce mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane. Capillary electrophoresis (CE) was validated by pyrosequencing as reliable and inexpensive high throughput method for identification and quantitative characterization of TALEN mediated mutations. Targeted COMT mutations were identified by CE in up to 74 % of the lines. In different events 8-99 % of the wild type COMT were converted to mutant COMT as revealed by pyrosequencing. Mutation frequencies among mutant lines were positively correlated to lignin reduction. Events with a mutation frequency of 99 % displayed a 29-32 % reduction of the lignin content compared to non-transgenic controls along with significantly reduced S subunit content and elevated hemicellulose content. CE analysis displayed similar peak patterns between primary COMT mutants and their vegetative progenies suggesting that TALEN mediated mutations were faithfully transmitted to vegetative progenies. This is the first report on genome editing in sugarcane. The findings demonstrate that targeted mutagenesis can improve cell wall characteristics for production of lignocellulosic ethanol in crops with highly complex genomes.

  3. Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic, Synergistic Lepidopteran-larvicidal and Nematicidal Activities

    PubMed Central

    Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2015-01-01

    Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed. PMID:25678849

  4. Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

    PubMed

    Liu, Tingting; Yao, Ruolan; Zhao, Yucheng; Xu, Sheng; Huang, Chuanlong; Luo, Jun; Kong, Lingyi

    2017-01-01

    Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure-function relationships of this important family of proteins.

  5. Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities.

    PubMed

    Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2015-01-01

    Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi9602(35-459)) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi9602(35-459) using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.

  6. Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

    PubMed Central

    Han, Ruizhi; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Chen, Jian

    2013-01-01

    2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering. PMID:24077706

  7. Iterative saturation mutagenesis of -6 subsite residues in cyclodextrin glycosyltransferase from Paenibacillus macerans to improve maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid synthesis.

    PubMed

    Han, Ruizhi; Liu, Long; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-12-01

    2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to -7). In this study, iterative saturation mutagenesis (ISM) was performed on the -6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites-Y167, G179, G180, and N193-was first performed and revealed that four mutants-Y167S, G179R, N193R, and G180R-produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the -6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

  8. Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

    PubMed Central

    Liu, Tingting; Yao, Ruolan; Zhao, Yucheng; Xu, Sheng; Huang, Chuanlong; Luo, Jun; Kong, Lingyi

    2017-01-01

    Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure–function relationships of this important family of proteins. PMID:28144249

  9. Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin

    PubMed Central

    Gu, Jingmin; Feng, Yingang; Feng, Xin; Sun, Changjiang; Lei, Liancheng; Ding, Wei; Niu, Fengfeng; Jiao, Lianying; Yang, Mei; Li, Yue; Liu, Xiaohe; Song, Jun; Cui, Ziyin; Han, Dong; Du, Chongtao; Yang, Yongjun; Ouyang, Songying; Liu, Zhi-Jie; Han, Wenyu

    2014-01-01

    The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin. PMID:24831957

  10. Structural and biochemical characterization reveals LysGH15 as an unprecedented "EF-hand-like" calcium-binding phage lysin.

    PubMed

    Gu, Jingmin; Feng, Yingang; Feng, Xin; Sun, Changjiang; Lei, Liancheng; Ding, Wei; Niu, Fengfeng; Jiao, Lianying; Yang, Mei; Li, Yue; Liu, Xiaohe; Song, Jun; Cui, Ziyin; Han, Dong; Du, Chongtao; Yang, Yongjun; Ouyang, Songying; Liu, Zhi-Jie; Han, Wenyu

    2014-05-01

    The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.

  11. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells.

    PubMed Central

    Roilides, E; Munson, P J; Levine, A S; Dixon, K

    1988-01-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells. PMID:2851732

  12. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

    SciTech Connect

    Roilides, E.; Munson, P.J.; Levine, A.S.; Dixon, K.

    1988-09-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.

  13. Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues.

    PubMed

    Rasila, Tiina S; Vihinen, Mauno; Paulin, Lars; Haapa-Paananen, Saija; Savilahti, Harri

    2012-01-01

    MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

  14. Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis.

    PubMed

    Cao, Heping; Deterding, Leesa J; Venable, John D; Kennington, Elizabeth A; Yates, John R; Tomer, Kenneth B; Blackshear, Perry J

    2006-02-15

    Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption-ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)-tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.

  15. Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis

    PubMed Central

    Cao, Heping; Deterding, Leesa J.; Venable, John D.; Kennington, Elizabeth A.; Yates, John R.; Tomer, Kenneth B.; Blackshear, Perry J.

    2005-01-01

    Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption–ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)–tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines. PMID:16262601

  16. Revealing Transient Interactions between Phosphatidylinositol-specific Phospholipase C and Phosphatidylcholine--Rich Lipid Vesicles

    NASA Astrophysics Data System (ADS)

    Yang, Boqian; He, Tao; Grauffel, Cédric; Reuter, Nathalie; Roberts, Mary; Gershenson, Anne

    2013-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes transiently interact with target membranes. Previous fluorescence correlation spectroscopy (FCS) experiments showed that Bacillus thuringiensis PI-PLC specifically binds to phosphatidylcholine (PC)-rich membranes and preferentially interacts with unilamellar vesicles that show larger curvature. Mutagenesis studies combined with FCS measurements of binding affinity highlighted the importance of interfacial PI-PLC tyrosines in the PC specificity. All-atom molecular dynamics simulations of PI-PLC performed in the presence of a PC membrane indicate these tyrosines are involved in specific cation-pi interactions with choline headgroups. To further understand those transient interactions between PI-PLC and PC-rich vesicles, we monitor single fluorescently labeled PI-PLC proteins as they cycle on and off surface-tethered small unilamellar vesicles using total internal reflection fluorescent microscopy. The residence times on vesicles along with vesicle size information, based on vesicle fluorescence intensity, reveal the time scales of PI-PLC membrane interactions as well as the curvature dependence. The PC specificity and the vesicle curvature dependence of this PI-PLC/membrane interaction provide insight into how the interface modulates protein-membrane interactions. This work was supported by the National Institute of General Medical Science of the National Institutes of Health (R01GM060418).

  17. Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

    PubMed Central

    Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; Hajighasemi, Mahbod; Nocek, Boguslaw; Khusnutdinova, Anna N.; Brown, Greg; Glinos, Julia; Flick, Robert; Skarina, Tatiana; Chernikova, Tatyana N.; Yim, Veronica; Brüls, Thomas; Paslier, Denis Le; Yakimov, Michail M.; Joachimiak, Andrzej; Ferrer, Manuel; Golyshina, Olga V.; Savchenko, Alexei; Golyshin, Peter N.; Yakunin, Alexander F.

    2017-01-01

    Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools. PMID:28272521

  18. Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids.

    PubMed

    Everson, R B

    1986-08-01

    Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

  19. Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids

    SciTech Connect

    Everson, R.B.

    1986-08-01

    Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

  20. Excavating the Genome: Large Scale Mutagenesis Screening for the Discovery of New Mouse Models

    PubMed Central

    Sundberg, John P.; Dadras, Soheil S.; Silva, Kathleen A.; Kennedy, Victoria E.; Murray, Stephen A.; Denegre, James; Schofield, Paul N.; King, Lloyd E.; Wiles, Michael; Pratt, C. Herbert

    2016-01-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis. While not automated to the level of the physiological phenotyping, histopathology provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being developed. PMID:26551941

  1. Arenaviruses and lethal mutagenesis. Prospects for new ribavirin-based interventions.

    PubMed

    Moreno, Héctor; Grande-Pérez, Ana; Domingo, Esteban; Martín, Verónica

    2012-11-06

    Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments.

  2. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

    PubMed Central

    Dalbadie-McFarland, G; Cohen, L W; Riggs, A D; Morin, C; Itakura, K; Richards, J H

    1982-01-01

    We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant. Images PMID:6983070

  3. Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

    PubMed

    Breyer, Didier; Herman, Philippe; Brandenburger, Annick; Gheysen, Godelieve; Remaut, Erik; Soumillion, Patrice; Van Doorsselaere, Jan; Custers, René; Pauwels, Katia; Sneyers, Myriam; Reheul, Dirk

    2009-01-01

    In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

  4. Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

    PubMed

    Haruki, M; Oohashi, Y; Mizuguchi, S; Matsuo, Y; Morikawa, M; Kanaya, S

    1999-07-09

    Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.

  5. [Mutagenesis of the human histamine H1 receptor and design of new antihistamine agents].

    PubMed

    Differding, E; Gillard, M; Moguilevsky, N; Varsalona, F; Noyer, M; Daliers, J; Goldstein, S; Neuwels, M; Lassoie, M A; Guillaume, J P; Bascour, M; Bollen, A; Hénichart, J P

    1996-01-01

    The binding cavity of histamine and histamine antagonists is explored using site directed mutagenesis of the human histamine H1 receptor and the amino acids involved in ligand binding are identified. Whereas Asp107 and Phe199 are important for both agonists and antagonists, two additional amino acids (Asn198 and Trp103) are required for efficient histamine binding. The binding site of antagonists is best defined as resulting from a strong ionic bond to Asp107, an orthogonal interaction between one of the aromatic rings with Phe199, and probably a hydrophobic interaction between the second aromatic ring and the lipophilic amino acids of the upper part of TMIV and TMV. This is consistent with structure-activity data of most described antagonists.

  6. Chromium (VI) potentiates mutagenesis by sodium azide but not ethyl methanesulfonate

    SciTech Connect

    LaVelle, J.M.; Witmer, C.M.

    1984-01-01

    A fluctuation test using Salmonella typhimurium strain 1535 has been used in an experimental protocol to assess biological effects of interactions between chromium (VI), such as K/sub 2/CrO/sub 4/, and two DNA-damaging agents, ethyl methanesulfonate (EMS), and sodium azide. For the combination of K/sub 2/CrO/sub 4/ and NaN/sub 3/, the response was significantly greater than expected suggesting a possible potentiation of mutagenesis. The opposite (a less-than-additive response) was found for the K/sub 2/CrO/sub 4//EMS combination. Both effects were found to be dose related to the concentration of potassium chromate used. Toxicity of the compounds or their combinations to the bacterial could not explain the results.

  7. The hidden side of unstable DNA repeats: mutagenesis at a distance

    PubMed Central

    Shah, Kartik A.; Mirkin, Sergei M.

    2015-01-01

    Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e. deletions, duplications, inversions, translocations and more complex shuffles. More recently, it has become clear that inherently unstable DNA repeats dramatically elevate mutation rates in surrounding DNA segments and that these mutations can occur upto ten kilobases away from the repetitive tract, a phenomenon we call repeat-induced mutagenesis (RIM). This review describes experimental data that led to the discovery and characterization of RIM and discusses the molecular mechanisms that could account for this phenomenon. PMID:25956860

  8. Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy.

    PubMed

    Hackett, Perry B; Largaespada, David A; Switzer, Kirsten C; Cooper, Laurence J N

    2013-04-01

    Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR(+) T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease.

  9. Site-specific mutagenesis of the calcium-binding photoprotein aequorin

    PubMed Central

    Tsuji, Frederick I.; Inouye, Satoshi; Goto, Toshio; Sakaki, Yoshiyuki

    1986-01-01

    The luminescent protein aequorin from the jellyfish Aequoria victoria emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. In order to understand the mechanism of the reaction, a study of structure-function relationships was undertaken with respect to modifying certain of its amino acid residues. This was done by carrying out oligonucleotide-directed site-specific mutagenesis of apoaequorin cDNA and expressing the mutagenized cDNA in Escherichia coli. Amino acid substitutions were made at the three Ca2+-binding sites, the three cysteines, and a histidine in one of the hydrophobic regions. Subsequent assay of the modified aequorin showed that the Ca2+-binding sites, the cysteines, and probably the histidine all play a role in the bioluminescence reaction of aequorin. PMID:16593774

  10. Mutagenesis as a Tool in Plant Genetics, Functional Genomics, and Breeding

    PubMed Central

    Sikora, Per; Chawade, Aakash; Larsson, Mikael; Olsson, Johanna; Olsson, Olof

    2011-01-01

    Plant mutagenesis is rapidly coming of age in the aftermath of recent developments in high-resolution molecular and biochemical techniques. By combining the high variation of mutagenised populations with novel screening methods, traits that are almost impossible to identify by conventional breeding are now being developed and characterised at the molecular level. This paper provides a comprehensive overview of the various techniques and workflows available to researchers today in the field of molecular breeding, and how these tools complement the ones already used in traditional breeding. Both genetic (Targeting Induced Local Lesions in Genomes; TILLING) and phenotypic screens are evaluated. Finally, different ways of bridging the gap between genotype and phenotype are discussed. PMID:22315587

  11. Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis.

    PubMed

    Lindsay, Andrew J; McCaffrey, Mary W

    2004-07-30

    Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.

  12. Enhancement of astaxanthin production from Haematococcus pluvialis mutants by three-stage mutagenesis breeding.

    PubMed

    Wang, Ni; Guan, Bin; Kong, Qing; Sun, Han; Geng, Zhaoyan; Duan, Liangfei

    2016-10-20

    Haematococcus pluvialis was modified for higher astaxanthin production compatible with the superiorities of high biomass and high activity by three-stage mutagenesis breeding. UV irradiation mutants named UV11-4 made an increase on cell dry weight, but showed a longer growth circle than the wild type. On the basis of UV mutants, ethyl methane sulphonate (EMS) mutants E2-5 cut down the latent phase, brought forward and extended the logarithmic phase. The inhibitor diphenylamine (DPA) was employed to screen high-yield astaxanthin producer by the color change of colonies from green to red on solid medium. Via the contravariant cultivation, proliferation and transformation, the mutant DPA12-2 possessed an 1.7-fold astaxanthin production compared to the wild type, reaching 47.21±3.30mg/g dry cells.

  13. Inducible pathway is required for mutagenesis in Salmonella typhimurium LT2

    SciTech Connect

    Orrego, C.; Eisenstadt, E.

    1987-06-01

    UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA/sup +/ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate. Mitomycin C induction of the Phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead the authors deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.

  14. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  15. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.

    PubMed

    Sakane, Yuto; Sakuma, Tetsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

  16. [Measurement of mutagenesis to study the effects of chemical agents]. Final report, August 1, 1993--July 31, 1994

    SciTech Connect

    Puck, T.T.

    1994-12-31

    This is the final report of a study conducted at the Eleanor Roosevelt Institute for Cancer Research, Inc. This study looked at mutagenesis as a measurement of the effects of chemical agents. Topics discussed in this report include: development of a new theory for the role of lipids and lipoproteins in the interactions of macromolecules; the action of caffeine in synergizing mutagenesis of agents like ionizing radiation by inhibition of cellular repair processes which was incorporated into a rapid procedure for detection of mutagenicity with high sensitivity; quantitative theoretical analysis of the mutagenesis process in cells exposed to physical and chemical mutagenic agents; theoretical analysis was developed leading to the conclusion that the visible chromosomal lesions described will also include a significant proportion of point mutations; application of this methodology for meaningful measurement of mutagenesis to study the effects of chemical agents was begun; and investigation of the cell cytoskeleton`s effect of genome exposure operating in the course of the differentiation process.

  17. A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase.

    PubMed

    Chen, Hui; Wu, Jianping; Yang, Lirong; Xu, Gang

    2013-12-01

    A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.

  18. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    ERIC Educational Resources Information Center

    Giron, Maria D.; Salto, Rafael

    2011-01-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

  19. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

  20. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Ascomycetous fungus Sclerotinia sclerotiorum is a devastating pathogen capable of infecting more than 400 plant species including many economically important crops. In order to gain a better mechanistic understanding of its non-specific host-pathogen interactions, random mutagenesis through Agro...

  1. Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases.

    PubMed

    Kawai, Narudo; Ochiai, Hiroshi; Sakuma, Tetsushi; Yamada, Lixy; Sawada, Hitoshi; Yamamoto, Takashi; Sasakura, Yasunori

    2012-06-01

    Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

  2. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases.

    PubMed

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-03-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44-100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1(Δ7/Δ7) fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.

  3. Predicting Resistance by Mutagenesis: Lessons from 45 Years of MBC Resistance

    PubMed Central

    Hawkins, Nichola J.; Fraaije, Bart A.

    2016-01-01

    When a new fungicide class is introduced, it is useful to anticipate the resistance risk in advance, attempting to predict both risk level and potential mechanisms. One tool for the prediction of resistance risk is laboratory selection for resistance, with the mutational supply increased through UV or chemical mutagenesis. This enables resistance to emerge more rapidly than in the field, but may produce mutations that would not emerge under field conditions. The methyl benzimidazole carbamates (MBCs) were the first systemic single-site agricultural fungicides, and the first fungicides affected by rapid evolution of target-site resistance. MBC resistance has now been reported in over 90 plant pathogens in the field, and laboratory mutants have been studied in nearly 30 species. The most common field mutations, including β-tubulin E198A/K/G, F200Y and L240F, have all been identified in laboratory mutants. However, of 28 mutations identified in laboratory mutants, only nine have been reported in the field. Therefore, the predictive value of mutagenesis studies would be increased by understanding which mutations are likely to emerge in the field. Our review of the literature indicates that mutations with high resistance factors, and those found in multiple species, are more likely to be reported in the field. However, there are many exceptions, possibly due to fitness penalties. Whether a mutation occurred in the same species appears less relevant, perhaps because β-tubulin is highly conserved so functional constraints are similar across all species. Predictability of mutations in other target sites will depend on the level and conservation of constraints. PMID:27895632

  4. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    PubMed

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  5. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

    ABSTRACT Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation. PMID:26787828

  6. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

    PubMed Central

    Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling. PMID:26799494

  7. Protective effect of basil (Ocimum basilicum L.) against oxidative DNA damage and mutagenesis.

    PubMed

    Berić, Tanja; Nikolić, Biljana; Stanojević, Jasna; Vuković-Gacić, Branka; Knezević-Vukcević, Jelena

    2008-02-01

    Mutagenic and antimutagenic properties of essential oil (EO) of basil and its major constituent Linalool, reported to possess antioxidative properties, were examined in microbial tests. In Salmonella/microsome and Escherichia. coli WP2 reversion assays both derivatives (0.25-2.0 microl/plate) showed no mutagenic effect. Salmonella. typhimurium TA98, TA100 and TA102 strains displayed similar sensitivity to both basil derivatives as non-permeable E. coli WP2 strains IC185 and IC202 oxyR. Moreover, the toxicity of basil derivatives to WP2 strains did not depend on OxyR function. The reduction of t-BOOH-induced mutagenesis by EO and Linalool (30-60%) was obtained in repair proficient strains of the E. coli K12 assay (Nikolić, B., Stanojević, J., Mitić, D., Vuković-Gacić, B., Knezević-Vukcević, J., Simić, D., 2004. Comparative study of the antimutagenic potential of vitamin E in different E. coli strains. Mutat. Res. 564, 31-38), as well as in E. coli WP2 IC202 strain. EO and Linalool reduced spontaneous mutagenesis in mismatch repair deficient E. coli K12 strains (27-44%). In all tests, antimutagenic effect of basil derivatives was comparable with that obtained with model antioxidant vitamin E. Linalool and vitamin E induced DNA strand breaks in Comet assay on S. cerevisiae 3A cells, but at non-genotoxic concentrations (0.075 and 0.025 microg/ml, respectively) they reduced the number of H(2)O(2)-induced comets (45-70% Linalool and 80-93% vitamin E). Obtained results indicate that antigenotoxic potential of basil derivatives could be attributed to their antioxidative properties.

  8. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

    PubMed

    Olshefsky, Audrey; Shehata, Laila; Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.

  9. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  10. High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

    PubMed Central

    Groot-Kormelink, Paul J.; Ferrand, Sandrine; Kelley, Nicholas; Bill, Anke; Freuler, Felix; Imbert, Pierre-Eloi; Marelli, Anthony; Gerwin, Nicole; Sivilotti, Lucia G.; Miraglia, Loren; Orth, Anthony P.; Oakeley, Edward J.; Schopfer, Ulrich; Siehler, Sandra

    2016-01-01

    High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation. PMID:27649498

  11. Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus).

    PubMed

    He, Xiaozhen; Li, Jie; Long, Yong; Song, Guili; Zhou, Peiyong; Liu, Qiuxiang; Zhu, Zuoyan; Cui, Zongbin

    2013-10-01

    The success of gene transfer has been demonstrated in many of vertebrate species, whereas the efficiency of producing transgenic animals remains pretty low due to the random integration of foreign genes into a recipient genome. The Sleeping Beauty (SB) transposon is able to improve the efficiency of gene transfer in zebrafish and mouse, but its activity in tilapia (Oreochromis niloticus) has yet to be characterized. Herein, we demonstrate the potential of using the SB transposon system as an effective tool for gene transfer and insertional mutagenesis in tilapia. A transgenic construct pT2/tiHsp70-SB11 was generated by subcloning the promoter of tilapia heat shock protein 70 (tiHsp70) gene, the SB11 transposase gene and the carp β-actin gene polyadenylation signal into the second generation of SB transposon. Transgenic tilapia was produced by microinjection of this construct with in vitro synthesized capped SB11 mRNA. SB11 transposon was detected in 28.89 % of founders, 12.9 % of F1 and 43.75 % of F2. Analysis of genomic sequences flanking integrated transposons indicates that this transgenic tilapia line carries two copies of SB transposon, which landed into two different endogenous genes. Induced expression of SB11 gene after heat shock was detected using reverse transcription PCR in F2 transgenic individuals. In addition, the Cre/loxP system was introduced to delete the SB11 cassette for stabilization of gene interruption and bio-safety. These findings suggest that the SB transposon system is active and can be used for efficient gene transfer and insertional mutagenesis in tilapia.

  12. Iterative saturation mutagenesis: a powerful approach to engineer proteins by systematically simulating Darwinian evolution.

    PubMed

    Acevedo-Rocha, Carlos G; Hoebenreich, Sabrina; Reetz, Manfred T

    2014-01-01

    Iterative saturation mutagenesis (ISM) is a widely applicable and powerful strategy for the efficient directed evolution of enzymes. First, one or more amino acid positions from the chosen enzyme are assigned to multi-residue sites (i.e., groups of amino acids or "multisites"). Then, the residues in each multisite are mutated with a user-defined randomization scheme to all canonical amino acids or a reduced amino acid alphabet. Subsequently, the genes of chosen variants (usually the best but not necessarily) are used as templates for saturation mutagenesis at other multisites, and the process is repeated until the desired degree of biocatalyst improvement has been achieved. Addressing multisites iteratively results in a so-called ISM scheme or tree with various upward branches or pathways. The systematic character of ISM simulates in vitro the natural process of Darwinian evolution: variation (library creation), selection (library screening), and amplification (template chosen for the next round of randomization). However, the main feature of ISM that distinguishes it from other directed evolution methods is the systematic probing of a defined segment of the protein sequence space, as it has been shown that ISM is much more efficient in terms of biocatalyst optimization than random methods such as error-prone PCR. In addition, ISM trees have also shed light on the emergence of epistasis, thereby rationally improving the strategies for evolving better enzymes. ISM was developed to improve catalytic properties such as rate, substrate scope, stereo- and regioselectivity using the Combinatorial Active-site Saturation Test (CAST), as well as chemical and thermal stability employing the B-Factor Iterative Test (B-FIT). However, ISM can also be invoked to manipulate such protein properties as binding affinity among other possibilities, including protein-protein interactions. Herein, we provide general guidelines for ISM, using CAST as the case study in the quest to

  13. Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    PubMed Central

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44–100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1Δ7/Δ7 fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics. PMID:23288935

  14. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    PubMed

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-05

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.