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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. Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

    PubMed Central

    Stern, Ana L.; Burgos, Emmanuel; Salmon, Laurent; Cazzulo, Juan J.

    2006-01-01

    Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease. PMID:16981853

  3. 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.

  4. 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

  5. Insertional mutagenesis reveals progression genes and checkpoints in MYC/Runx2 lymphomas

    PubMed Central

    2008-01-01

    In this study we have exploited the power of insertional mutagenesis to elucidate tumor progression pathways in mice carrying two oncogenes (MYC/Runx2) that collaborate to drive early lymphoma development. Neonatal infection of these mice with Moloney murine leukemia virus (MLV) resulted in accelerated tumor onset with associated increases in clonal complexity and lymphoid dissemination. Large-scale analysis of retroviral integration sites in these tumors revealed a profound bias towards a narrow range of target genes including Jdp2 (Jundm2), D cyclin and Pim family genes. Remarkably, direct PCR analysis of integration hot-spots revealed that every progressing tumor consisted of multiple clones harbouring hits at these loci, giving access to large numbers of independent insertion events and uncovering the contrasting mutagenic mechanisms operating at each target gene. Direct PCR analysis showed that high frequency targeting occurs only in the tumor environment in vivo and is specific for the progression gene set. These results indicate that early lymphomas in MYC/Runx2 mice remain dependent on exogenous growth signals and that progression can be achieved by constitutive activation of pathways converging on a cell cycle checkpoint that acts as the major rate-limiting step for lymphoma outgrowth. PMID:17545590

  6. 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…

  7. 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…

  8. 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

  9. 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

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

    PubMed

    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

    2016-08-18

    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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations*

    PubMed Central

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

    2014-01-01

    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: Ser68, Ser47, Thr62/Ile66, Thr56, Thr32/Gly45, and Met52. 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 Asp65–Ser68 that form a major part of the lining of the pore. PMID:24436330

  19. 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.

  20. 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.

  1. Genome-wide mutagenesis reveals that ORF7 is a novel VZV skin-tropic factor.

    PubMed

    Zhang, Zhen; Selariu, Anca; Warden, Charles; Huang, Grace; Huang, Ying; Zaccheus, Oluleke; Cheng, Tong; Xia, Ningshao; Zhu, Hua

    2010-07-01

    The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. Mutagenesis of tGCN5 core region reveals two critical surface residues F90 and R140

    SciTech Connect

    Mehta, Kinjal Rajesh; Chan, Yan M.; Lee, Man X.; Yang, Ching Yao; Voloshchuk, Natalya; Montclare, Jin Kim

    2010-09-24

    Research highlights: {yields} Mutagenesis of the tGCN5 core region reveals two residues important for function. {yields} Developed a fluorescent lysate-based activity assay to assess mutants. {yields} Surface-exposed residues F90 and R140 of tGCN5 are critical for H3 acetylation. -- Abstract: Tetrahymena General Control Non-Derepressor 5 (tGCN5) is a critical regulator of gene transcription via acetylation of histones. Since the acetylation ability has been attributed to the 'core region', we perform mutagenesis of residues within the tGCN5 'core region' in order to identify those critical for function and stability. Residues that do not participate in catalysis are identified, mutated and characterized for activity, structure and thermodynamic stability. Variants I107V, Q114L, A121T and A130S maintain the acetylation function relative to wild-type tGCN5, while variants F90Y, F112R and R140H completely abolish function. Of the three non-functional variants, since F112 is mutated into a non-homologous charged residue, a loss in function is expected. However, the remaining two variants are mutated into homologous residues, suggesting that F90 and R140 are critical for the activity of tGCN5. While mutation to homologous residue maintains acetylation of histone H3 for the majority of the variants, the two surface-exposed residues, F90 and R140, appear to be essential for tGCN5 function, structure or stability.

  7. 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

  8. Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis.

    PubMed

    Gaffaney, Jon D; Dunning, F Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R

    2008-11-14

    Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC50 for Ca2+-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

  9. Genome-wide mutagenesis of dengue virus reveals plasticity of the NS1 protein and enables generation of infectious tagged reporter viruses.

    PubMed

    Eyre, Nicholas S; Johnson, Stephen M; Eltahla, Auda A; Aloi, Maria; Aloia, Amanda L; McDevitt, Christopher A; Bull, Rowena A; Beard, Michael R

    2017-09-27

    Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and sub-tropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and employed next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that regions within capsid, NS1 and the 3' UTR were most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high resolution imaging of its localization to the surface and interior of viral replication vesicles. Additionally, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed

  10. 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

  11. 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

  12. 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

  13. Hiding personal information reveals the worst.

    PubMed

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

    2016-01-26

    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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Whole exome sequencing reveals a functional mutation in the GAIN domain of the Bai2 receptor underlying a forward mutagenesis hyperactivity QTL.

    PubMed

    Speca, David J; Trimmer, James S; Peterson, Andrew S; Díaz, Elva

    2017-09-12

    The identification of novel genes underlying complex mouse behavioral traits remains an important step in understanding normal brain function and its dysfunction in mental health disorders. To identify dominant mutations that influence locomotor activity, we performed a mouse N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen and mapped several loci as quantitative traits. Here we describe the fine-mapping and positional cloning of a hyperactivity locus mapped to the medial portion of mouse chromosome four. We employed a modified recombinant progeny testing approach to fine-map the confidence interval from ≈20 Mb down to ≈5 Mb. Whole exome resequencing of all exons in this region revealed a single missense mutation in the adhesion G protein-coupled receptor brain-specific angiogenesis inhibitor 2 (Bai2). This mutation, R619W, is located in a critical extracellular domain that is a hotspot for mutations in this receptor class. We find that in two different mammalian cell lines, surface expression of Bai2 R619W is markedly reduced relative to wild-type Bai2, suggesting that R619W is a loss-of-function mutation. Our results highlight the powerful combination of ENU mutagenesis and next-generation sequencing to identify specific mutations that manifest as subtle behavioral phenotypes.

  20. Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

    PubMed

    Ramsey, Meghan E; Bender, Tobias; Klimowicz, Amy K; Hackett, Kathleen T; Yamamoto, Ami; Jolicoeur, Adrienne; Callaghan, Melanie M; Wassarman, Karen M; van der Does, Chris; Dillard, Joseph P

    2015-09-01

    Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion. © 2015 John Wiley & Sons Ltd.

  1. 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

  2. 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.

  3. 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

  4. Molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase.

    PubMed

    de la Torre, Fernando; Moya-García, Aurelio A; Suárez, María-Fernanda; Rodríguez-Caso, Carlos; Cañas, Rafael A; Sánchez-Jiménez, Francisca; Cánovas, Francisco M

    2009-04-01

    We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the "open-to-closed" transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.

  5. 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.

  6. Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

    PubMed Central

    Horton, Lori B.; Shanker, Sreejesh; Mikulski, Rose; Brown, Nicholas G.; Phillips, Kevin J.; Lykissa, Ernest; Venkataram Prasad, B. V.

    2012-01-01

    Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward β-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 μM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable. PMID:22908171

  7. Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

    PubMed Central

    Huser, Camille A.; Gilroy, Kathryn L.; de Ridder, Jeroen; Kilbey, Anna; Borland, Gillian; Mackay, Nancy; Jenkins, Alma; Bell, Margaret; Herzyk, Pawel; van der Weyden, Louise; Adams, David J.; Rust, Alistair G.; Cameron, Ewan; Neil, James C.

    2014-01-01

    Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer

  8. 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

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

    PubMed Central

    Du, Lawrence; Tracy, Sharon

    2016-01-01

    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 -527 bp 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. PMID:26896592

  10. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

    PubMed

    Jiao, Yang; Guo, Rongxian; Tang, Peipei; Kang, Xilong; Yin, Junlei; Wu, Kaiyue; Geng, Shizhong; Li, Qiuchun; Sun, Jun; Xu, Xiulong; Zhou, Xiaohui; Gan, Junji; Jiao, Xinan; Liu, Xiufan; Pan, Zhiming

    2017-03-03

    Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 10(6.6) -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Mutagenesis of N-terminal residues of feline foamy virus Gag reveals entirely distinct functions during capsid formation, particle assembly, Gag processing and budding.

    PubMed

    Liu, Yang; Betts, Matthew J; Lei, Janet; Wei, Guochao; Bao, Qiuying; Kehl, Timo; Russell, Robert B; Löchelt, Martin

    2016-08-22

    Foamy viruses (FVs) of the Spumaretrovirinae subfamily are distinct retroviruses, with many features of their molecular biology and replication strategy clearly different from those of the Orthoretroviruses, such as human immunodeficiency, murine leukemia, and human T cell lymphotropic viruses. The FV Gag N-terminal region is responsible for capsid formation and particle budding via interaction with Env. However, the critical residues or motifs in this region and their functional interaction are currently ill-defined, especially in non-primate FVs. Mutagenesis of N-terminal Gag residues of feline FV (FFV) reveals key residues essential for either capsid assembly and/or viral budding via interaction with the FFV Env leader protein (Elp). In an in vitro Gag-Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Gradient sedimentation analyses of cytosolic proteins indicate that wild-type Gag is mostly assembled into virus capsids. Moreover, proteolytic processing of Gag correlates with capsid assembly and is mostly, if not completely, independent from particle budding. In addition, Gag processing correlates with the presence of packaging-competent FFV genomic RNA suggesting that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. Though an appended heterogeneous myristoylation signal rescues Gag particle budding of mutants unable to form capsids or defective in interacting with Elp, it fails to generate infectious particles that co-package Pol, as evidenced by a lack of Gag processing. Changes in proteolytic Gag processing, intracellular capsid assembly, particle budding and infectivity of defined N-terminal Gag mutants highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. Discussion of these findings will be based on a recent model developed for Gag

  2. 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

  3. 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.

  4. 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.

  5. Lethal mutagenesis of viruses.

    PubMed

    Perales, Celia; Martín, Verónica; Domingo, Esteban

    2011-11-01

    Lethal mutagenesis aims at extinguishing viruses by increased mutagenesis prompted by virus-specific mutagenic agents, mainly nucleoside analogues. It is derived from the error threshold relationship of quasispecies theory, and it is slowly finding its way towards a clinical application. We summarize the current situation of research in this field of antiviral therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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.

  8. Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced iron release from human serum transferrin

    PubMed Central

    Steere, Ashley N.; Chasteen, N. Dennis; Miller, Brendan F.; Smith, Valerie C.; MacGillivray, Ross T.A.; Mason, Anne B.

    2012-01-01

    The recent crystal structure of two monoferric human serum transferrin (FeNhTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details of this binding interaction which dictates iron delivery to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two FeNhTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca2+ binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of iron release at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of a H318A sTFR mutant allows assignment of a small pH dependent initial decrease in the fluorescent signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated iron release from the C-lobe of the Fe2hTF/sTFR Δ757–760 complex. The loss is accounted for by the inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs in order to promote receptor-stimulated iron release from the C-lobe of hTF. PMID:22356162

  9. Chemical and UV Mutagenesis.

    PubMed

    Bose, Jeffrey L

    2016-01-01

    The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

  10. Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin.

    PubMed

    Steere, Ashley N; Chasteen, N Dennis; Miller, Brendan F; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2012-03-13

    The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.

  11. 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

  12. Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli.

    PubMed

    Zhang, Huibin; Susanto, Teodorus T; Wan, Yue; Chen, Swaine L

    2016-04-12

    Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5' UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.

  13. A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

    PubMed

    Toogood, Helen S; Fryszkowska, Anna; Hulley, Martyn; Sakuma, Michiyo; Mansell, David; Stephens, Gill M; Gardiner, John M; Scrutton, Nigel S

    2011-03-21

    We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

    PubMed

    Kudalkar, Shalley N; Njuma, Olive J; Li, Yongjiang; Muldowney, Michelle; Fuanta, N Rene; Goodwin, Douglas C

    2015-03-03

    Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

  20. Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

    PubMed Central

    2014-01-01

    Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational

  1. Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila.

    PubMed

    Hollfelder, Dominik; Frasch, Manfred; Reim, Ingolf

    2014-06-17

    The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/

  2. 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.

  3. 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)

  4. 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)

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

    PubMed

    Dreux, Marlène; Dao Thi, Viet Loan; Fresquet, Judith; Guérin, Maryse; Julia, Zélie; Verney, Géraldine; Durantel, David; Zoulim, Fabien; Lavillette, Dimitri; Cosset, François-Loïc; Bartosch, Birke

    2009-02-01

    HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Individual olfactory perception reveals meaningful nonolfactory genetic information

    PubMed Central

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

    2015-01-01

    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. PMID:26100865

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. Mariner-based transposon mutagenesis for Bacteroides species.

    PubMed

    Ichimura, Minoru; Uchida, Keiko; Nakayama-Imaohji, Haruyuki; Hirakawa, Hideki; Tada, Tomoyo; Morita, Hidetoshi; Yasutomo, Koji; Okazaki, Katsuichiro; Kuwahara, Tomomi

    2014-06-01

    Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. 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

  2. Information Processing Limitations as Revealed by Temporal Discrimination

    ERIC Educational Resources Information Center

    Lavoie, Philippe; Grondin, Simon

    2004-01-01

    Information processing limit is a fundamental issue in cognitive psychology. One particular way of studying it is to adopt a temporal span perspective. In this experiment, Weber fractions based on thresholds for duration discrimination are used for adopting this perspective. The results showed that, contrary to the constant predicted by Weber's…

  3. 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

  4. 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.

  5. Environmental stress induces trinucleotide repeat mutagenesis in human cells.

    PubMed

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

    2015-03-24

    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.

  6. 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

  7. Empirical complexities in the genetic foundations of lethal mutagenesis.

    PubMed

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

    2013-10-01

    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.

  8. Deciding to Reveal Sexual Information and Sexuality Education in Mother-Daughter Relationships

    ERIC Educational Resources Information Center

    Coffelt, Tina A.

    2017-01-01

    Communication privacy management theory informed this study of nine mothers and their 18- or 19-year-old daughters who were interviewed to understand privacy rule foundations that influence their decisions to reveal or conceal sexual information. Findings reveal the salience of motivation and the risk-benefit ratio when making decisions about…

  9. What's Worth Talking About? Information Theory Reveals How Children Balance Informativeness and Ease of Production.

    PubMed

    Bannard, Colin; Rosner, Marla; Matthews, Danielle

    2017-07-01

    Of all the things a person could say in a given situation, what determines what is worth saying? Greenfield's principle of informativeness states that right from the onset of language, humans selectively comment on whatever they find unexpected. In this article, we quantify this tendency using information-theoretic measures and report on a study in which we tested the counterintuitive prediction that children will produce words that have a low frequency given the context, because these will be most informative. Using corpora of child-directed speech, we identified adjectives that varied in how informative (i.e., unexpected) they were given the noun they modified. In an initial experiment ( N = 31) and in a replication ( N = 13), 3-year-olds heard an experimenter use these adjectives to describe pictures. The children's task was then to describe the pictures to another person. As the information content of the experimenter's adjective increased, so did children's tendency to comment on the feature that adjective had encoded. Furthermore, our analyses suggest that children balance informativeness with a competing drive to ease production.

  10. 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 work...

  11. 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...

  12. 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…

  13. 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…

  14. 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

  15. 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.

  16. 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.

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

    PubMed Central

    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. PMID:24273524

  18. 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...

  19. 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 §...

  20. Theory of lethal mutagenesis for viruses.

    PubMed

    Bull, J J; Sanjuán, R; Wilke, C O

    2007-03-01

    Mutation is the basis of adaptation. Yet, most mutations are detrimental, and elevating mutation rates will impair a population's fitness in the short term. The latter realization has led to the concept of lethal mutagenesis for curing viral infections, and work with drugs such as ribavirin has supported this perspective. As yet, there is no formal theory of lethal mutagenesis, although reference is commonly made to Eigen's error catastrophe theory. Here, we propose a theory of lethal mutagenesis. With an obvious parallel to the epidemiological threshold for eradication of a disease, a sufficient condition for lethal mutagenesis is that each viral genotype produces, on average, less than one progeny virus that goes on to infect a new cell. The extinction threshold involves an evolutionary component based on the mutation rate, but it also includes an ecological component, so the threshold cannot be calculated from the mutation rate alone. The genetic evolution of a large population undergoing mutagenesis is independent of whether the population is declining or stable, so there is no runaway accumulation of mutations or genetic signature for lethal mutagenesis that distinguishes it from a level of mutagenesis under which the population is maintained. To detect lethal mutagenesis, accurate measurements of the genome-wide mutation rate and the number of progeny per infected cell that go on to infect new cells are needed. We discuss three methods for estimating the former. Estimating the latter is more challenging, but broad limits to this estimate may be feasible.

  1. 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.

  2. 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

  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. 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.

  5. GATMD: γ-Aminobutyric Acid Transporter Mutagenesis Database

    PubMed Central

    Anderson, Cynthia M.; Kidd, Patrick D.; Eskandari, Sepehr

    2010-01-01

    Since the cloning of the first γ-aminobutyric acid (GABA) transporter (GAT1; SLC6A1) from rat brain in 1990, more than 50 published studies have provided structure–function information on investigator-designed rat and mouse GAT1 mutants. To date, more than 200 of 599 GAT1 residues have been subjected to mutagenesis experiments by substitution with different amino acids, and the resulting transporter functional properties have significantly advanced our understanding of the mechanism of Na+- and Cl–-coupled GABA transport by this important member of the neurotransmitter:sodium symporter family. Moreover, many studies have addressed the functional consequences of amino acid deletion or insertion at various positions along the primary sequence. The enormity of this growing body of structure–function information has prompted us to develop GABA Transporter Mutagenesis Database (GATMD), a web-accessible, relational database of manually annotated biochemical, functional and pharmacological data reported on GAT1—the most intensely studied GABA transporter isoform. As of the last update of GATMD, 52 GAT1 mutagenesis papers have yielded 3360 experimental records, which collectively contain a total of ∼100 000 annotated parameters. Database URL: http://physiology.sci.csupomona.edu/GATMD/ PMID:21131297

  6. Lethal mutagenesis in viruses and bacteria.

    PubMed

    Chen, Peiqiu; Shakhnovich, Eugene I

    2009-10-01

    In this work we study how mutations that change physical properties of cell proteins (stability) affect population survival and growth. We present a model in which the genotype is presented as a set folding free energies of cell proteins. Mutations occur upon replication, so stabilities of some proteins in daughter cells differ from those in the parent cell by amounts deduced from the distribution of mutational effects on protein stability. The genotype-phenotype relationship posits that the cell's fitness (replication rate) is proportional to the concentration of its folded proteins and that unstable essential proteins result in lethality. Simulations reveal that lethal mutagenesis occurs at a mutation rate close to seven mutations in each replication of the genome for RNA viruses and at about half that rate for DNA-based organisms, in accord with earlier predictions from analytical theory and experimental results. This number appears somewhat dependent on the number of genes in the organisms and the organism's natural death rate. Further, our model reproduces the distribution of stabilities of natural proteins, in excellent agreement with experiments. We find that species with high mutation rates tend to have less stable proteins compared to species with low mutation rates.

  7. Facilitators on networks reveal optimal interplay between information exchange and reciprocity.

    PubMed

    Szolnoki, Attila; Perc, Matjaž; Mobilia, Mauro

    2014-04-01

    Reciprocity is firmly established as an important mechanism that promotes cooperation. An efficient information exchange is likewise important, especially on structured populations, where interactions between players are limited. Motivated by these two facts, we explore the role of facilitators in social dilemmas on networks. Facilitators are here mirrors to their neighbors-they cooperate with cooperators and defect with defectors-but they do not participate in the exchange of strategies. As such, in addition to introducing direct reciprocity, they also obstruct information exchange. In well-mixed populations, facilitators favor the replacement and invasion of defection by cooperation as long as their number exceeds a critical value. In structured populations, on the other hand, there exists a delicate balance between the benefits of reciprocity and the deterioration of information exchange. Extensive Monte Carlo simulations of social dilemmas on various interaction networks reveal that there exists an optimal interplay between reciprocity and information exchange, which sets in only when a small number of facilitators occupy the main hubs of the scale-free network. The drawbacks of missing cooperative hubs are more than compensated for by reciprocity and, at the same time, the compromised information exchange is routed via the auxiliary hubs with only marginal losses in effectivity. These results indicate that it is not always optimal for the main hubs to become leaders of the masses, but rather to exploit their highly connected state to promote tit-for-tat-like behavior.

  8. Facilitators on networks reveal optimal interplay between information exchange and reciprocity

    NASA Astrophysics Data System (ADS)

    Szolnoki, Attila; Perc, Matjaž; Mobilia, Mauro

    2014-04-01

    Reciprocity is firmly established as an important mechanism that promotes cooperation. An efficient information exchange is likewise important, especially on structured populations, where interactions between players are limited. Motivated by these two facts, we explore the role of facilitators in social dilemmas on networks. Facilitators are here mirrors to their neighbors—they cooperate with cooperators and defect with defectors—but they do not participate in the exchange of strategies. As such, in addition to introducing direct reciprocity, they also obstruct information exchange. In well-mixed populations, facilitators favor the replacement and invasion of defection by cooperation as long as their number exceeds a critical value. In structured populations, on the other hand, there exists a delicate balance between the benefits of reciprocity and the deterioration of information exchange. Extensive Monte Carlo simulations of social dilemmas on various interaction networks reveal that there exists an optimal interplay between reciprocity and information exchange, which sets in only when a small number of facilitators occupy the main hubs of the scale-free network. The drawbacks of missing cooperative hubs are more than compensated for by reciprocity and, at the same time, the compromised information exchange is routed via the auxiliary hubs with only marginal losses in effectivity. These results indicate that it is not always optimal for the main hubs to become leaders of the masses, but rather to exploit their highly connected state to promote tit-for-tat-like behavior.

  9. Whole-exome sequencing studies of parathyroid carcinomas reveal novel PRUNE2 mutations, distinctive mutational spectra related to APOBEC-catalyzed DNA mutagenesis and mutational enrichment in kinases associated with cell migration and invasion.

    PubMed

    Yu, Willie; McPherson, John R; Stevenson, Mark; van Eijk, Ronald; Heng, Hong Lee; Newey, Paul; Gan, Anna; Ruano, Dina; Huang, Dachuan; Poon, Song Ling; Ong, Choon Kiat; van Wezel, Tom; Cavaco, Branca; Rozen, Steven G; Tan, Patrick; Teh, Bin T; Thakker, Rajesh V; Morreau, Hans

    2015-02-01

    Cell division cycle 73 (CDC73), encoding the protein parafibromin, is the most prevalent mutated gene in familial and sporadic parathyroid carcinoma (PC). To identify additional genetic abnormalities in PCs. Whole-exome sequencing was performed using DNA from seven pairs of matched PCs and one triplet containing double primary tumor and normal leukocyte. Somatic variants were confirmed using Sanger sequencing and recurrently mutated genes were assessed in 13 additional PCs as well as 40 parathyroid adenomas (PA). PC had an average of 51 somatic variants/tumor (range 3-176) with approximately 58% of variants occurring as nonsynonymous single nucleotide variants. The importance of CDC73 in PC is reinforced with a remarkable preferential amplification of the mutant CDC73 allele. Furthermore, recurrent germ line and somatic mutations in prune homolog 2 [Drosophila] (PRUNE2) were found in PC and computationally predicted to be deleterious; in addition, recurrent mutations in kinase genes related to cell migration and invasion were found. PRUNE2 showed recurrent mutations in 18% (4/22) of PCs with additional screening in 40 PAs revealing only one rare missense polymorphism (Asp1677Asn). For the first time, the mutational signature associated with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-catalyzed cytosine-to-uracil deamination is found in a subset of PC. This study outlines the genetic landscape of PC and attempts to characterize the mutational processes shaping the PC genome.

  10. [Stress-induced cellular adaptive mutagenesis].

    PubMed

    Zhu, Linjiang; Li, Qi

    2014-04-01

    The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

  11. 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

  12. 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.

  13. Highly Efficient Targeted Mutagenesis in Mice Using TALENs

    PubMed Central

    Panda, Sudeepta Kumar; Wefers, Benedikt; Ortiz, Oskar; Floss, Thomas; Schmid, Bettina; Haass, Christian; Wurst, Wolfgang; Kühn, Ralf

    2013-01-01

    Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. PMID:23979585

  14. A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping

    PubMed Central

    Mason, Anne B.; Byrne, Shaina L.; Everse, Stephen J.; Roberts, Samantha E.; Chasteen, N. Dennis; Smith, Valerie C.; MacGillivray, Ross T. A.; Kandemir, Banu; Bou-Abdallah, Fadi

    2015-01-01

    Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR. PMID:19693784

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Economical analysis of saturation mutagenesis experiments

    PubMed Central

    Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  20. 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.

  1. 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

  2. Genetic code evolution reveals the neutral emergence of mutational robustness, and information as an evolutionary constraint.

    PubMed

    Massey, Steven E

    2015-04-24

    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 between organisms, a

  3. P53 Gene Mutagenesis in Breast Cancer

    DTIC Science & Technology

    2005-03-01

    suppressor gene in sporadic breast tumours . 1991. Loss of chromosome 17 pl3 sequences and mutation of p53 Oncogene 5 :1573-1579. in human breast...COVERED March 2005 Final (I Aug 2000 - 1 Feb 2004) 4. TITLE AND SUBTITLE 5 . FUNDING NUMBERS p53 Gene Mutagenesis in Breast Cancer DAMD17-00-1-0204 6. AUTHOR...The central hypothesis of this proposal is that variability in the patterns of p 5 3 mutagenesis in breast cancer reflects differences in exposures to

  4. 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

  5. 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…

  6. 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…

  7. 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...

  8. 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...

  9. 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.

  10. Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits.

    PubMed

    Lerner, Talia N; Shilyansky, Carrie; Davidson, Thomas J; Evans, Kathryn E; Beier, Kevin T; Zalocusky, Kelly A; Crow, Ailey K; Malenka, Robert C; Luo, Liqun; Tomer, Raju; Deisseroth, Karl

    2015-07-30

    Recent progress in understanding the diversity of midbrain dopamine neurons has highlighted the importance--and the challenges--of defining mammalian neuronal cell types. Although neurons may be best categorized using inclusive criteria spanning biophysical properties, wiring of inputs, wiring of outputs, and activity during behavior, linking all of these measurements to cell types within the intact brains of living mammals has been difficult. Here, using an array of intact-brain circuit interrogation tools, including CLARITY, COLM, optogenetics, viral tracing, and fiber photometry, we explore the diversity of dopamine neurons within the substantia nigra pars compacta (SNc). We identify two parallel nigrostriatal dopamine neuron subpopulations differing in biophysical properties, input wiring, output wiring to dorsomedial striatum (DMS) versus dorsolateral striatum (DLS), and natural activity patterns during free behavior. Our results reveal independently operating nigrostriatal information streams, with implications for understanding the logic of dopaminergic feedback circuits and the diversity of mammalian neuronal cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. An APOBEC Cytidine Deaminase Mutagenesis Pattern is Widespread in Human Cancers

    PubMed Central

    Roberts, Steven A.; Lawrence, Michael S.; Klimczak, Leszek J.; Grimm, Sara A.; Fargo, David; Stojanov, Petar; Kiezun, Adam; Kryukov, Gregory V.; Carter, Scott L.; Saksena, Gordon; Harris, Shawn; Shah, Ruchir R.; Resnick, Michael A.; Getz, Gad; Gordenin, Dmitry A.

    2013-01-01

    Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome datasets conformed to stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes of 14 cancer types, mostly from TCGA, revealed significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2E subtype was clearly enriched with tumors displaying the APOBEC mutation pattern, suggesting this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC mutagenesis is carcinogenic. PMID:23852170

  12. 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.

  13. Genetic analysis of mutagenesis in aging Escherichia coli colonies.

    PubMed

    Taddei, F; Halliday, J A; Matic, I; Radman, M

    1997-10-01

    Bacteria live in unstructured and structured environments, experiencing feast and famine lifestyles. Bacterial colonies can be viewed as model structured environments. SOS induction and mutagenesis have been observed in aging Escherichia coli colonies, in the absence of exogenous sources of DNA damage. This cAMP-dependent mutagenesis occurring in Resting Organisms in a Structured Environment (ROSE) is unaffected by a umuC mutation and therefore differs from both targeted UV mutagenesis and recA730 (SOS constitutive) untargeted mutagenesis. As a recB mutation has only a minor effect on ROSE mutagenesis it also differs from both adaptive reversion of the lacI33 allele and from iSDR (inducible Stable DNA Replication) mutagenesis. Besides its recA and lexA dependence, ROSE mutagenesis is also uvrB and polA dependent. These genetic requirements are reminiscent of the untargeted mutagenesis in lambda phage observed when unirradiated lambda infects UV-irradiated E. coli. These mutations, which are not observed in aging liquid cultures, accumulate linearly with the age of the colonies. ROSE mutagenesis might offer a good model for bacterial mutagenesis in structured environments such as biofilms and for mutagenesis of quiescent eukaryotic cells.

  14. High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

    PubMed Central

    Delacôte, Fabien; Perez, Christophe; Guyot, Valérie; Duhamel, Marianne; Rochon, Christelle; Ollivier, Nathalie; Macmaster, Rachel; Silva, George H.; Pâques, Frédéric; Daboussi, Fayza; Duchateau, Philippe

    2013-01-01

    Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells. PMID:23359797

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Translesion DNA Synthesis and Mutagenesis in Eukaryotes

    PubMed Central

    Sale, Julian E.

    2013-01-01

    The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells. PMID:23457261

  6. 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

  7. Social anxiety and anger identification: bubbles reveal differential use of facial information with low spatial frequencies.

    PubMed

    Langner, Oliver; Becker, Eni S; Rinck, Mike

    2009-06-01

    We investigated the facial information that socially anxious and nonanxious individuals utilize to judge emotions. Using a reversed-correlation technique, we presented participants with face images that were masked with random bubble patterns. These patterns determined which parts of the face were visible in specific spatial-frequency bands. This masking allowed us to establish which locations and spatial frequencies were helping participants to successfully discriminate angry faces from neutral ones. Although socially anxious individuals performed as well as nonanxious individuals on the emotion-discrimination task, they did not utilize the same facial information for the task. The fine details (high spatial frequencies) around the eyes were discriminative for both groups, but only socially anxious participants additionally processed rough configural information (low spatial frequencies).

  8. 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.

  9. Revealing representational content with pattern-information fMRI—an introductory guide

    PubMed Central

    Bandettini, Peter A.; Kriegeskorte, Nikolaus

    2009-01-01

    Conventional statistical analysis methods for functional magnetic resonance imaging (fMRI) data are very successful at detecting brain regions that are activated as a whole during specific mental activities. The overall activation of a region is usually taken to indicate involvement of the region in the task. However, such activation analysis does not consider the multivoxel patterns of activity within a brain region. These patterns of activity, which are thought to reflect neuronal population codes, can be investigated by pattern-information analysis. In this framework, a region's multivariate pattern information is taken to indicate representational content. This tutorial introduction motivates pattern-information analysis, explains its underlying assumptions, introduces the most widespread methods in an intuitive way, and outlines the basic sequence of analysis steps. PMID:19151374

  10. 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.

  11. 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.

  12. 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

  13. Multivoxel Pattern Analysis Reveals 3D Place Information in the Human Hippocampus

    PubMed Central

    Jeffery, Kate J.

    2017-01-01

    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 2D 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 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 (HC) expressed place information that was sensitive to changes along both horizontal and vertical axes. This is suggestive of isotropic 3D place encoding. In 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 HC 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 STATEMENT The spatial world is 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 horizontal and vertical information is equally well represented. Here, we measured fMRI response patterns while participants moved within a virtual 3D

  14. 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 (c) 2016 APA, all rights reserved).

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

    PubMed

    Clarke, Alex

    2015-04-21

    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.

  16. Eye-Movements Reveal Attention to Social Information in Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Fletcher-Watson, S.; Leekam, S. R.; Benson, V.; Frank, M. C.; Findlay, J. M.

    2009-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental condition in which children show reduced attention to social aspects of the environment. However in adults with ASD, evidence for social attentional deficits is equivocal. One problem is that many paradigms present social information in an unrealistic, isolated way. This study presented adults…

  17. 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

  18. 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

  19. Probabilistic analysis of recessive mutagenesis screen strategies.

    PubMed

    Silver, Jeremy D; Hilton, Douglas J; Bahlo, Melanie; Kile, Benjamin T

    2007-01-01

    Random mutagenesis screens for recessive phenotypes require three generations of breeding, using either a backcross (BC) or intercross (IC) strategy. Hence, they are more costly and technically demanding than those for dominant phenotypes. Maximizing the return from these screens requires maximizing the number of mutations that are bred to homozyosity in the G(3) generation. Using a probabilistic approach, we compare different designs of screens for recessive phenotypes and the impact each one has on the number of mutations that can be effectively screened. We address the issue of BC versus IC strategies and consider genome-wide, region-specific screens and suppressor screens. We find that optimally designed BC and IC screens allow the screening of, on average, similar numbers of mutations but that interpedigree variation is more pronounced when the IC strategy is employed. By conducting a retrospective analysis of published mutagenesis screens, we show that, depending on the strategy, a threefold difference in the numbers of mutations screened per animal used could be expected. This method allows researchers to contrast, for a range of experimental designs, the cost per mutation screened and to maximize the number of mutations that one can expect to screen in a given experiment.

  20. Lethal mutagenesis in a structured environment.

    PubMed

    Steinmeyer, Shelby H; Wilke, Claus O

    2009-11-07

    We analyze how lethal mutagenesis operates in a compartmentalized host. We assume that different compartments receive different amounts of mutagen and that virions can migrate among compartments. We address two main questions: (1) To what extent can refugia, i.e., compartments that receive little mutagen, prevent extinction? (2) Does migration among compartments limit the effectiveness of refugia? We find that if there is little migration, extinction has to be achieved separately in all compartments. In this case, the total dose of mutagen administered to the host needs to be so high that the mutagen is effective even in the refugia. By contrast, if migration is extensive, then lethal mutagenesis is effective as long as the average growth in all compartments is reduced to below replacement levels. The effectiveness of migration is governed by the ratio of virion replication and death rates, R(0). The smaller R(0), the less migration is necessary to neutralize refugia and the less mutagen is necessary to achieve extinction at high migration rates.

  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. 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.

  3. 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.

  4. Causal visual interactions as revealed by an information theoretic measure and fMRI.

    PubMed

    Hinrichs, H; Heinze, H J; Schoenfeld, M A

    2006-07-01

    In the present study, we evaluated the direction of the effective connectivity between fMRI activations in neural structures mediating preserved visual function in a patient with homonymous hemianopsia due to a posterior cerebral artery stroke. Although the lesion affected the primary visual cortex, the visual abilities of this patient included above-chance verbal reports of movement and color change as well as the discrimination of movement direction in his hemianopic field. These abilities were coupled with awareness (Riddoch syndrome). The strength and the direction of the interactions between visual regions were assessed by applying directed transinformation (T), a nonparametric information theoretic causal measure sensitive to linear as well as to nonlinear interactions. In the healthy hemisphere, T identified a strong flow of information from visual area V1 to V5 during stimulation by visual movement and from V1 to V4/V8 during stimulation by color change. In addition, during color change stimulation, a bi-directional flow was observed between V4/V8 and V5, suggesting crosstalk between these regions. In the lesioned hemisphere, the color change stimulation evoked a stronger flow from V5 to V4/V8 and a flow from V4/V8 to V2. These observations provide support for the hypothesis that visual information is mediated via subcortical pathways that bypass V1 and project first to higher-tier visual areas V5 and V4/V8 then subsequently to lower-tier area V2.

  5. 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

  6. 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.

  7. Trans-Saccadic Repetition Priming: ERPs Reveal On-line Integration of Information across Words

    PubMed Central

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

    2015-01-01

    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 & 2) were compared with priming effects obtained with centrally located targets (Experiment 3). In Experiment 2, target stimuli were preceded by a 100 ms 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. PMID:26656872

  8. 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.

  9. 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

  10. 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).

  11. An information theoretic framework reveals a tunable allosteric network in group II chaperonins.

    PubMed

    Lopez, Tom; Dalton, Kevin; Tomlinson, Anthony; Pande, Vijay; Frydman, Judith

    2017-09-01

    ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

  12. 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

  13. 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

  14. 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.

  15. Magnetoencephalography reveals altered auditory information processing in youth with obsessive-compulsive disorder.

    PubMed

    Korostenskaja, Milena; Harris, Elana; Giovanetti, Cathy; Horn, Paul; Wang, Yingying; Rose, Douglas; Fujiwara, Hisako; Xiang, Jing

    2013-05-30

    Patients with obsessive-compulsive disorder (OCD) often report sensory intolerances which may lead to significant functional impairment. This study used auditory evoked fields (AEFs) to address the question of whether neural correlates of sensory auditory information processing differ in youth with OCD compared with healthy comparison subjects (HCS). AEFs, recorded with a whole head 275-channel magnetoencephalography system, were elicited in response to binaural auditory stimuli from 10 pediatric subjects with OCD (ages 8-13, mean 11 years, 6 males) and 10 age- and gender-matched HCS. Three major neuromagnetic responses were studied: M70 (60-80 ms), M100 (90-120 ms), and M150 (130-190 ms). When compared with HCS, subjects with OCD demonstrated delayed latency of the M100 response. In subjects with OCD the amplitude of the M100 and M150 responses was significantly greater in the right hemisphere compared with the left hemisphere. Current results suggest that when compared with HCS, subjects with OCD have altered auditory information processing, evident from the delayed latency of the M100 response, which is thought to be associated with the encoding of physical stimulus characteristics. Interhemispheric asymmetry with increased M100 and M150 amplitudes over the right hemisphere compared with the left hemisphere was found in young OCD subjects. These results should be interpreted with caution due to the high variability rate of responses in both HCS and OCD subjects, as well as the possible effect of medication in OCD subjects. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. 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

  17. 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

  18. 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. © 2014 ARVO.

  19. 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

  20. Temporal Information Partition Networks (TIPNets): A Process Network Framework to Reveal Eco-Hydrologic Feedbacks, Responses and Shifts

    NASA Astrophysics Data System (ADS)

    Goodwell, A. E.; Kumar, P.

    2016-12-01

    Within an ecosystem, components of the atmosphere, vegetation, and the root-soil system participate in forcing and feedback reactions at varying time scales and intensities. These interactions constitute a complex network that exhibits behavioral shifts due to perturbations ranging from weather events to long-term drought or land use change. However, it is challenging to characterize this shifting network due to multiple drivers, non-linear interactions, and feedback-induced synchronization. To overcome these issues, we implement a process network approach where eco-hydrologic time-series variables are nodes and information measures are links. We introduce a Temporal Information Partition Network (TIPNet) framework in which multivariate lagged mutual information between pairs of source nodes and target nodes is decomposed into synergistic, redundant, and unique components, each of which reveals different aspects of interactions within the network. We use methods to compute information measures given as few as 200 data points to construct TIPNets based on 1-minute weather station data (radiation Rg, air temperature Ta, wind speed WS, relative humidity RH, precipitation PPT, and leaf wetness LWet) from the Intensively Managed Critical Zone Observatory (IML-CZO) during the growing season of 2015. We find that the source node pairs (Ta, Rg) and (Ta, RH) tend to be synchronized sources that provide dominantly redundant information to target nodes during the day and night, respectively. Meanwhile, source node pairs that include WS often provide synergistic information, indicating the presence of multiple non-redundant influences. We assess temporal shifts in network behavior for weather conditions including rainy periods, dew, and accumulating drought to show how process networks reveal ecosystem behaviors and responses.

  1. Early information processing contributions to object individuation revealed by perception of illusory figures.

    PubMed

    Naughtin, Claire K; Mattingley, Jason B; Dux, Paul E

    2016-12-01

    To isolate multiple coherent objects from their surrounds, each object must be represented as a stable perceptual entity across both time and space. Recent theoretical and empirical work has proposed that this process of object individuation is a mid-level operation that emerges around 200-300 ms after stimulus onset. However, this hypothesis is based on paradigms that have potentially obscured earlier effects. Furthermore, no study to date has directly assessed whether object individuation occurs for task-irrelevant objects. In the present study we used electroencephalography (EEG) to measure the time course of individuation, for stimuli both within and outside the focus of attention, to assess the information processing stage at which object individuation arises for both types of objects. We developed a novel paradigm involving items defined by illusory contours, which allowed us to vary the number of to-be-individuated objects while holding the physical elements of the display constant (a design characteristic not present in earlier work). As early as 100 ms after stimulus onset, event-related potentials tracked the number of objects in the attended hemifield, but not those in the unattended hemifield. By contrast, both attended and unattended objects could be individuated at a later stage. Our findings challenge recent conceptualizations of the time course of object individuation and suggest that this process arises earlier for attended than unattended items, implying that voluntary spatial attention influences the time course of this operation. Copyright © 2016 the American Physiological Society.

  2. Use of local statistics to reveal hidden information of pollution hotspots in urban soil geochemistry

    NASA Astrophysics Data System (ADS)

    Zhang, Chaosheng

    2017-04-01

    The identification of pollution hotspots is an important approach for a better understanding of spatial distribution patterns and the exploration for their influencing factors in environmental studies. One of the most often asked questions in an environmental investigation is: Where are the pollution hotspots? This presentation explains one of the popularly used methodologies called local index of spatial association (LISA) and its applications in urban geochemical studies in Galway, Ireland and London of the UK. The LISA is a useful tool for identifying pollution hotspots and classifying them into spatial clusters and spatial outliers. The results were affected by the definition of weight function, data transformation and existence of extreme values, and it is suggested that all these influencing factors should be considered until reasonable and reliable results are obtained. This method has been applied to identify Pb pollution in Galway, polluted areas in bonfires sites, elevated P and REE concentrations in London. Hotspots in identified in urban soils are related to locations of high road density, traditional festival bonfires, industries and other human activities. The results of hotspots analysis provide useful information for the management of urban soils.

  3. 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.

  4. Medial prefrontal aberrations in major depressive disorder revealed by cytoarchitectonically informed voxel-based morphometry

    PubMed Central

    Bludau, Sebastian; Bzdok, Danilo; Gruber, Oliver; Kohn, Nils; Riedl, Valentin; Sorg, Christian; Palomero-Gallagher, Nicola; Müller, Veronika I.; Hoffstaedter, Felix; Amunts, Katrin; Eickhoff, Simon B.

    2017-01-01

    Objective The heterogeneous human frontal pole has been identified as a node in the dysfunctional network of major depressive disorder. The contribution of the medial (socio-affective) versus lateral (cognitive) frontal pole to major depression pathogenesis is currently unclear. The present study performs morphometric comparison of the microstructurally informed subdivisions of human frontal pole between depressed patients and controls using both uni- and multivariate statistics. Methods Multi-site voxel- and region-based morphometric MRI analysis of 73 depressed patients and 73 matched controls without psychiatric history. Frontal pole volume was first compared between depressed patients and controls by subdivision-wise classical morphometric analysis. In a second approach, frontal pole volume was compared by subdivision-naive multivariate searchlight analysis based on support vector machines. Results Subdivision-wise morphometric analysis found a significantly smaller medial frontal pole in depressed patients with a negative correlation of disease severity and duration. Histologically uninformed multivariate voxel-wise statistics provided converging evidence for structural aberrations specific to the microstructurally defined medial area of the frontal pole in depressed patients. Conclusions Across disparate methods, we demonstrated subregion specificity in the left medial frontal pole volume in depressed patients. Indeed, the frontal pole was shown to structurally and functionally connect to other key regions in major depression pathology like the anterior cingulate cortex and the amygdala via the uncinate fasciculus. Present and previous findings consolidate the left medial portion of the frontal pole as particularly altered in major depression. PMID:26621569

  5. 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.

  6. 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.

  7. 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

  8. Proteomics informed by transcriptomics reveals Hendra virus sensitizes bat cells to TRAIL-mediated apoptosis.

    PubMed

    Wynne, James W; Shiell, Brian J; Marsh, Glenn A; Boyd, Victoria; Harper, Jennifer A; Heesom, Kate; Monaghan, Paul; Zhou, Peng; Payne, Jean; Klein, Reuben; Todd, Shawn; Mok, Lawrence; Green, Diane; Bingham, John; Tachedjian, Mary; Baker, Michelle L; Matthews, David; Wang, Lin-Fa

    2014-01-01

    Bats are a major reservoir of emerging infectious viruses. Many of these viruses are highly pathogenic to humans however bats remain asymptomatic. The mechanism by which bats control viral replication is unknown. Here we utilize an integrated approach of proteomics informed by transcriptomics to compare the response of immortalized bat and human cells following infection with the highly pathogenic bat-borne Hendra virus (HeV). The host response between the cell lines was significantly different at both the mRNA and protein levels. Human cells demonstrated minimal response eight hours post infection, followed by a global suppression of mRNA and protein abundance. Bat cells demonstrated a robust immune response eight hours post infection, which led to the up-regulation of apoptosis pathways, mediated through the tumor necrosis factor-related apoptosis inducing ligand (TRAIL). HeV sensitized bat cells to TRAIL-mediated apoptosis, by up-regulating death receptor transcripts. At 48 and 72 hours post infection, bat cells demonstrated a significant increase in apoptotic cell death. This is the first study to comprehensively compare the response of bat and human cells to a highly pathogenic zoonotic virus. An early induction of innate immune processes followed by apoptosis of virally infected bat cells highlights the possible involvement of programmed cell death in the host response. Our study shows for the first time a side-by-side high-throughput analysis of a dangerous zoonotic virus in cell lines derived from humans and the natural bat host. This enables a way to search for divergent mechanisms at a molecular level that may influence host pathogenesis.

  9. 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.

  10. Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

    NASA Astrophysics Data System (ADS)

    Gao, Guang-Yu; Li, Yu; Wang, Wei; Wang, Shu-Feng; Dongping, Zhong; Gong, Qi-Huang

    2015-01-01

    Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe, tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule’s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity. Project supported by the National Basic Research Program of China (Grant Nos. 2013CB921904, 2009CB930504, and 2013CB328700) and the National Natural Science Foundation of China (Grant Nos. 11074016, 11121091, 10934001, 61177020, 11134001, and 10828407).

  11. Targeted mutagenesis of an odorant receptor co-receptor using TALEN in Ostrinia furnacalis.

    PubMed

    Yang, Bin; Fujii, Takeshi; Ishikawa, Yukio; Matsuo, Takashi

    2016-03-01

    Genome editing using transcription activator-like effector nuclease (TALEN) has been applied for various model organisms but not yet for agricultural pest insects. In this study, TALEN-mediated mutagenesis of the gene encoding odorant receptor co-receptor (Orco) of an important agricultural pest Ostrinia furnacalis (OfurOrco) was carried out. Of the two pairs of TALEN constructs designed, one generated somatic and germline mutations at rates of 70.8% and 20.8%, respectively. Physiological and behavioral analyses using a gas chromatograph-electroantennographic detector system and a wind tunnel, respectively, revealed that antennal responses to sex pheromone components were decreased to trace levels, and behavioral responses were abolished in OfurOrco mutants. This study demonstrated that TALEN-mediated mutagenesis is applicable to pest insects, and these results will open the way for a better understanding of chemosensory systems in wild insects. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Systematic Dissection and Trajectory-Scanning Mutagenesis of the Molecular Interface That Ensures Specificity of Two-Component Signaling Pathways

    PubMed Central

    Lubin, Emma A.; Ashenberg, Orr; Skerker, Jeffrey M.; Laub, Michael T.

    2010-01-01

    Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk. PMID:21124821

  13. Germline mutagenesis mediated by Sleeping Beauty transposon system in mice

    PubMed Central

    Takeda, Junji; Keng, Vincent W; Horie, Kyoji

    2007-01-01

    Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice. PMID:18047691

  14. 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.

  15. 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.

  16. 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

  17. History of attempts to quantify environmental mutagenesis

    SciTech Connect

    Hollaender, A.

    1981-01-01

    It became obvious in the early 1960's that the ready recognition of mutations produced by chemicals could have a profound influence on the refinement of methods to detect environmental mutagens. The experience derived over the previous 30 years in characterizing the effects of ionizing and ultraviolet radiation on the genetic mechanism came to serve us in good stead. Although the effects of chemicals are considerably more complicated and often require the analysis of individual substances, nonetheless, the area has developed rapidly in recent decades. The establishment and historical background of the International Association of Environmental Mutagen Societies (IAEMS) will be discussed. An attempt at the quantitation of chemical effects has been developed in comparison with radiation mutagenesis. As a first step, a definition of the Mutagen Burden or unavoidable exposure to chemicals will be discussed. A mathematical approach (Haynes/Eckhardt) will be considered and finally an outline for the comprehensive investigation of detailed interscience study will be made of less than six chemicals.

  18. Genomic approaches to DNA repair and mutagenesis.

    PubMed

    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. 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.

  20. 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). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. 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

  2. Genomic Approaches to DNA repair and Mutagenesis

    PubMed Central

    Wyrick, John J.; Roberts, Steven A.

    2015-01-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. PMID:26411877

  3. Herpesvirus mutagenesis facilitated by infectious bacterial artificial chromosomes (iBACs).

    PubMed

    Robinson, Karl E; Mahony, Timothy J

    2015-01-01

    A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction.

  4. 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

  5. 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.

  6. 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)

  7. 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

  8. 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

  9. Treating the Einstein-Hilbert action as a higher derivative Lagrangian: revealing the missing information about conformal non-invariance

    NASA Astrophysics Data System (ADS)

    Nikolić, Branislav

    2017-08-01

    The Hamiltonian formulation of conformally invariant Weyl-squared higher derivative theory teaches us that conformal symmetry is expressed through particular first class constraints related to the absence of the three-metric determinant and the trace of the extrinsic curvature from the theory. Any term depending on them which is added to this theory breaks conformal invariance and turns these constraints into second class ones. Such second class constraints are missing in the standard canonical formulation of the conformally non-invariant Einstein-Hilbert theory. It is demonstrated that such constraints do appear if the theory is treated as a higher derivative one: if the extrinsic curvature is promoted to an independent variable, the apparently missing information about conformal behavior is revealed.

  10. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

    PubMed

    Genovesi, Laura A; Ng, Ching Ging; Davis, Melissa J; Remke, Marc; Taylor, Michael D; Adams, David J; Rust, Alistair G; Ward, Jerrold M; Ban, Kenneth H; Jenkins, Nancy A; Copeland, Neal G; Wainwright, Brandon J

    2013-11-12

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.

  11. 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

  12. 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.

  13. Transposon Tn916 mutagenesis in Bacillus anthracis.

    PubMed Central

    Ivins, B E; Welkos, S L; Knudson, G B; Leblanc, D J

    1988-01-01

    Mutagenesis of Bacillus anthracis by the streptococcal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tcr) transconjugants were obtained at a frequency of 1.6 X 10(-8) per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S.faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10(-5). A Tcr B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10(-4) and 5.8 X 10(-6), respectively. The transfer of Tn916 occurred only on membrane filters, since no Tcr transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tcr B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tcr transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan. Images PMID:2826334

  14. 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

  15. Socially anxious people reveal more personal information with virtual counselors that talk about themselves using intimate human back stories.

    PubMed

    Kang, Sin-Hwa; Gratch, Jonathan

    2012-01-01

    In this paper, we describe our findings from research designed to explore the effect of virtual human counselors' self-disclosure using intimate human back stories on real human clients' social responses in psychological counseling sessions. To investigate this subject, we designed an experiment involving two conditions of the counselors' self-disclosure: human back stories and computer back stories. We then measured socially anxious users' verbal self-disclosure. The results demonstrated that highly anxious users revealed personal information more than less anxious users when they interacted with virtual counselors who disclosed intimate information about themselves using human back stories. Furthermore, we found that greater inclination toward facilitated self-disclosure from highly anxious users following interaction with virtual counselors who employed human back stories rather than computer back stories. In addition, a further analysis of socially anxious users' feelings of rapport demonstrated that virtual counselors elicited more rapport with highly anxious users than less anxious users when interacting with counselors who employed human back stories. This outcome was not found in the users' interactions with counselors who employed computer back stories.

  16. 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

  17. 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

  18. 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.

  19. USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

    PubMed

    Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

    2015-12-15

    Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Stabilization of a prokaryotic LAT transporter by random mutagenesis.

    PubMed

    Rodríguez-Banqueri, Arturo; Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Kowalczyk, Lukasz; Perálvarez-Marín, Alex; Palacín, Manuel; Vázquez-Ibar, José Luis

    2016-04-01

    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. © 2016 Rodríguez-Banqueri et al.

  1. 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

  2. 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.

  3. Fluorescent protein engineering by in vivo site-directed mutagenesis

    PubMed Central

    Ceballos, Melvys Valledor; Hu, Qinghua; Schiller, Paul; Myers, Richard S.

    2012-01-01

    Summary In vivo site-directed mutagenesis by ssDNA recombineering is a facile method to change the color of fluorescent proteins without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the E. coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the fluorescent protein. Eight different fluorescent proteins (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. PMID:22639380

  4. 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.

  5. Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

    NASA Astrophysics Data System (ADS)

    Yao, Risheng; Li, Manman; Deng, Shengsong; Hu, Huajia; Wang, Huai; Li, Fenghe

    2012-04-01

    Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of Trichoderma viride was developed by ultraviolet (UV) and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain Trichoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.

  6. 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.

  7. Production and Screening of High Yield Avermectin B1b Mutant of Streptomyces avermitilis 41445 Through Mutagenesis

    PubMed Central

    Siddique, Samia; Syed, Quratulain; Adnan, Ahmad; Qureshi, Fahim Ashraf

    2014-01-01

    Background: Secondary metabolite production from wild strains is very low for economical purpose therefore certain strain improvement strategies are required to achieve hundred times greater yield of metabolites. Most important strain improvement techniques include physical and chemical mutagenesis. Broad spectrum mutagenesis through UV irradiation is the most important and convenient physical method. Objectives: The present study was conducted for enhanced production of avermectin B1b from Streptomyces avermitilis 41445 by mutagenesis using ultraviolet (UV) radiation, ethidium bromide (EB), and ethyl methanesulfonate (EMS) as mutagens. Materials and Methods: S. avermitilis DSM 41445 maintained on yeast extract malt extract glucose medium (YMG) was used as inoculum for SM2 fermentation medium. Spores of S. avermitilis DSM 41445 were exposed to UV radiation for physical broad spectrum mutagenesis and to EMS and EB for chemical mutagenesis. For each mutagen, the lethality rate and mutation rate were calculated along with positive mutation rate. Results: Avermectin B1b-hyper-producing mutant, produced using these three different methods, was selected according to the HPLC results. The mutant obtained after 45 minutes of UV radiation to the spores of S. avermitilis 41445, was found to be the best mutant for the enhanced production of avermectin B1b component (254.14 mg/L). Other avermectin B1b-hyper-producing mutants, were obtained from EMS (1 µL/mL) and EB (30 µL/mL) treatments, and yielded 202.63 mg/L and 199.30 mg/L of B1b, respectively. Conclusions: The hereditary stability analysis of the UV mentioning 45 minutes revealed the UV exposure time for mutants and 3 represented the colony taken from the plate irradiated for 45 minutes mutant showed that the production of avermectin B1b remained constant and no reverse mutation occurred after 15 generations. PMID:25147669

  8. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. © The Author(s).

  9. Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli.

    PubMed

    Ohta, T; Watanabe, K; Moriya, M; Shirasu, Y; Kada, T

    1983-02-01

    Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.

  10. 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…

  11. Now and future of mouse mutagenesis for human disease models.

    PubMed

    Gondo, Yoichi

    2010-09-01

    One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to develop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution strains and collaborative cross strains are also available to elucidate the complex traits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human. Copyright © 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  12. 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

  13. 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.

  14. Insertional mutagenesis using Tnt1 retrotransposon in potato

    USDA-ARS?s Scientific Manuscript database

    Potato is the third most important food crop in the world. However, genetics and genomics research of potato has lagged behind many major crop species due to its autotetraploidy and a highly heterogeneous genome. Insertional mutagenesis using T-DNA or transposable elements, which is available in sev...

  15. 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…

  16. 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

  17. Targeted mutagenesis using CRISPR/Cas in inbred potatoes

    USDA-ARS?s Scientific Manuscript database

    Targeted mutagenesis using sequence-specific nucleases (SSNs) has been well established in several important crop species, but is in need of improvement in potato (Solanum tuberosum L.). For over a century, potatoes have been bred as autotetraploids (2n = 4x = 48), relying on F1 selections and clona...

  18. 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.

  19. Illegitimate recombination: an efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis.

    PubMed

    Khattak, Faisal Asghar; Kumar, Ashutosh; Kamal, Elisabeth; Kunisch, Ralph; Lewin, Astrid

    2012-09-11

    The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.

  20. Statistical evidence for a biochemical pathway of natural, sequence-targeted G/C to C/G transversion mutagenesis in Haemophilus influenzae Rd.

    PubMed

    Merkl, R; Fritz, H J

    1996-11-01

    Markov chain analysis of the Haemophilus influenzae Rd genome reveals striking under-representation of three palindromic tetranucleotide strings (CCGG, GGCC and CATG), accompanied by over-representation of six tetranucleotide strings that are derived from the former by exchanging strand location of the two residues making up a G/C nucleotide pair at the terminal palindrome position. Constraints are outlined for a molecular model able to explain the phenomenon as the result of sequence-targeted, enzyme-driven G/C to C/G transversion mutagenesis. Possible participation in the process by components of known DNA mismatch repair or restriction/modification systems (in particular, cytosine methylation) is discussed. The effect widens the spectrum of enzyme-driven, specific mutagenesis beyond the formerly described C/G to T/A transition (VSP repair of Escherichia coli). Potential evolutionary benefits of enzymatic pathways of specific mutagenesis can be envisioned.

  1. 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

  2. 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

  3. 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.

  4. Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

    PubMed

    Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

    2017-04-01

    To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

  5. 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

  6. Insight into Binding of Phosphodiesterase-9A Selective Inhibitors by Crystal Structures and Mutagenesis

    SciTech Connect

    Wang, H.; Luo, X; Ye, M; Hou, J; Robinson, H; Ke, H

    2010-01-01

    PDE9 inhibitors have been studied as therapeutics for treatment of cardiovascular diseases, diabetes, and neurodegenerative disorders. To illustrate the inhibitor selectivity, the crystal structures of the PDE9A catalytic domain in complex with the enantiomers of PDE9 inhibitor 1-(2-chlorophenyl)-6-(3,3,3-trifluoro-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-one ((R)-BAY73-6691 or (S)-BAY73-6691, 1r or 1s) were determined and mutagenesis was performed. The structures showed that the fluoromethyl groups of 1r and 1s had different orientations while the other parts of the inhibitors commonly interacted with PDE9A. These differences may explain the slightly different affinity of 1r (IC{sub 50} = 22 nM) and 1s (IC{sub 50} = 88 nM). The mutagenesis experiments revealed that contribution of the binding residues to the inhibitor sensitivity varies dramatically, from few-fold to 3 orders of magnitude. On the basis of the crystal structures, a hypothesized compound that simulates the recently published PDE9 inhibitors was modeled to provide insight into the inhibitor selectivity.

  7. 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.

  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. 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.

  11. 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.

  12. Nonrandom mutagenesis. Progress report, March 1, 1981-February 28, 1982

    SciTech Connect

    Goldsby, R.A.

    1981-01-01

    The ultimate goal is the development of tools, approaches and systems which will increase our ability to detect and control mutagenesis. We have continued to develop hybrid cell lines suited to the investigation of the expression and mutagenesis of human cell surface markers. The development and characterization of the monoclonal antibody probes to identify and characterize variation in selected human cell surface antigens has continued. Human X mouse T lymphoma hybrids have proven valuable in obtaining clonal populations expressing cell surface determinants characteristic of particular differentiated cell types. We have constructed a set of human lymphocyte X mouse T lymphoma hybrids which have proven useful for the mapping of cell surface determinants to particular chromosomes.

  13. European Community research on environmental mutagenesis and carcinogenesis.

    PubMed Central

    Sors, A I

    1993-01-01

    Within the 12 Member States of the European Community (EC), environmental policy is now formulated primarily at Community level. As a result, the EC has important regulatory responsibilities for the protection of workers, consumers, and the general public from risks that may arise from environmental chemicals, foremost among them potential carcinogens and mutagens. An important part of EC environmental research and development is intended to provide a scientific basis for these regulations as well as increasing understanding of the basic mechanisms involved in environmental carcinogenesis and mutagenesis. This paper contains a brief introduction to EC environment policy and research, followed by an overview of EC chemicals control activities that are of particular relevance to the research and development program. Community-level research on environmental mutagenesis and carcinogenesis is then reviewed in some detail, including the achievements of recent projects, the scientific content of the current program, and perspectives for the future. PMID:8143645

  14. 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

  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. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  16. 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.

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

    PubMed

    Portelli, Geoffrey; Barrett, John M; Hilgen, Gerrit; Masquelier, Timothée; Maccione, Alessandro; Di Marco, Stefano; Berdondini, Luca; Kornprobst, Pierre; Sernagor, Evelyne

    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.

  18. 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

  19. Environmental mutagenesis and radiation biology: The legacy of William Morgan.

    PubMed

    Schwartz, Jeffrey L

    2017-07-25

    A symposium entitled Environmental Mutagenesis and Radiation Biology was held on September 27, 2016 to honor the memory of Dr. William F. Morgan who passed away unexpectedly on November 13, 2015. The speakers presented the latest reviews on homologous recombination repair, induced genetic instability, bystander effects, and risk estimate development. Their presentations are presented following the introduction. Copyright © 2017. Published by Elsevier B.V.

  20. Transcriptional mutagenesis: causes and involvement in tumor development

    PubMed Central

    Brégeon, Damien; Doetsch, Paul W.

    2013-01-01

    The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

  1. 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

  2. ENU Mutagenesis Identifies Mice with Morbid Obesity and Severe Hyperinsulinemia Caused by a Novel Mutation in Leptin

    PubMed Central

    Cheng, Chih-Ya; Chou, Chuan-Kai; Jheng, Huei-Fen; Chuang, You-Chung; Yang, Chia-Ning; Lin, Ya-Tzu; Hsu, Chih-Wei; Cheng, Irene H.; Chen, Shiow-Yi; Tsai, Shih-Jen; Liou, Ying-Jay; Tsai, Yau-Sheng

    2010-01-01

    Background Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals. Methodology/Principal Findings To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin145E/145E mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin145E/145E mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin145E/145E mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region. Conclusions/Significance Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of

  3. SOMA: a single oligonucleotide mutagenesis and cloning approach.

    PubMed

    Pfirrmann, Thorsten; Lokapally, Ashwin; Andréasson, Claes; Ljungdahl, Per; Hollemann, Thomas

    2013-01-01

    Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

  4. 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.

  5. Mutagenesis by the autoxidation of iron with isolated DNA

    SciTech Connect

    Loeb, L.A.; James, E.A.; Waltersdorph, A.M.; Klebanoff, S.J. )

    1988-06-01

    Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes. These radicals damage cellular constituents and have been casually implicated in the pathogenesis of many human diseases. The authors report here that oxygen free radicals generated by Fe{sup 2+} in aqueous solution are mutagenic. Aerobic incubation of {phi}X174 am3 (amber 3 mutation) DNA with Fe{sup 2+} results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts. Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold. Both killing and mutagenesis can be prevented by binding of Fe{sup 2+} with deferoxamine or by the addition of catalase or mannitol. These results suggest that DNA damage and mutagenesis brought about by Fe{sup 2+} are likely to occur by a Fenton-type mechanism. DNA sequence analysis of the Fe{sup 2+}-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine. These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.

  6. 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.

  7. Factors affecting recall of different types of personal genetic information about Alzheimer's disease risk: the REVEAL study.

    PubMed

    Besser, Andria G; Sanderson, Saskia C; Roberts, J Scott; Chen, Clara A; Christensen, Kurt D; Lautenbach, Denise M; Cupples, L Adrienne; Green, Robert C

    2015-01-01

    Data were obtained through a multisite clinical trial in which different types of genetic risk-related information were disclosed to individuals (n = 246) seeking a risk assessment for Alzheimer's disease. Six weeks after disclosure, 83% of participants correctly recalled the number of risk-increasing APOE alleles they possessed, and 74% correctly recalled their APOE genotype. While 84% of participants recalled their lifetime risk estimate to within 5 percentage points, only 51% correctly recalled their lifetime risk estimate exactly. Correct recall of the number of APOE risk-increasing alleles was independently associated with higher education (p < 0.001), greater numeracy (p < 0.05) and stronger family history of Alzheimer's disease (p < 0.05). Before adjustments for confounding, correct recall of APOE genotype was also associated with higher education, greater numeracy and stronger family history of Alzheimer's disease, as well as with higher comfort with numbers and European American ethnicity (all p < 0.05). Correct recall of the lifetime risk estimate was independently associated only with younger age (p < 0.05). Recall of genotype-specific information is high, but recall of exact risk estimates is lower. Incorrect recall of numeric risk may lead to distortions in understanding risk. Further research is needed to determine how best to communicate different types of genetic risk information to patients, particularly to those with lower educational levels and lower numeracy. Health-care professionals should be aware that each type of genetic risk information may be differentially interpreted and retained by patients and that some patient subgroups may have more problems with recall than others. © 2015 S. Karger AG, Basel.

  8. Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis.

    PubMed

    Yokoyama, Keiichi; Utsumi, Hiroe; Nakamura, Takefumi; Ogaya, Daisuke; Shimba, Nobuhisa; Suzuki, Eiichiro; Taguchi, Seiichi

    2010-08-01

    Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 A space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.

  9. Bridging the gap between molecular and elemental mass spectrometry: higher energy collisional dissociation (HCD) revealing elemental information.

    PubMed

    Esteban-Fernández, Diego; El-Khatib, Ahmed H; Moraleja, Irene; Gómez-Gómez, M Milagros; Linscheid, Michael W

    2015-02-03

    Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS.

  10. EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine.

    PubMed

    Acanda, Yosvanis; Martínez, Óscar; Prado, María Jesús; González, María Victoria; Rey, Manuel

    2014-03-01

    Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration. Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.

  11. 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.

  12. Different colors reveal different information: how nutritional stress affects the expression of melanin- and structurally based ornamental plumage.

    PubMed

    McGraw, Kevin J; Mackillop, Emiko A; Dale, James; Hauber, Mark E

    2002-12-01

    Avian plumage colors have emerged recently as model systems for investigating the types of information that can be signaled by showy sexual displays in animals. In many species, the brightness of carotenoid-based plumage reflects the health and condition of individuals and is used in mate selection. The information contained in melanin-based and structurally based ornamental colors in birds is less well resolved, however. We subjected male house sparrows Passer domesticus and brown-headed cowbirds Molothrus ater to stressful nutritional conditions during molt to test the hypothesis that melanin- and structurally based plumage colors are nutritionally condition-dependent. We restricted food access for treatment males during randomized 6 h periods on 4 days per week, while allowing control birds access to food ad libitum throughout the course of the molt. We found that the size and brightness of the melanin-based throat badges in male house sparrows were not affected by nutritional stress. Similarly, there were no differences between treatment and control male cowbirds in the size or brightness of the melanin-based brown hood. However, the structurally based iridescent plumage of cowbirds was indicative of the nutritional condition of males during molt. Nutritionally stressed cowbirds grew significantly less colorful plumage than did males with access to food ad libitum. These results are consistent with observations in other avian species that different types of plumage color communicate different sets of information. Melanin ornaments are less sensitive to nutritional conditions during molt and instead may reflect the hormonal status and/or competitive ability of males, whereas structural coloration appears to be an accurate signal of health and condition.

  13. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  14. 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.

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

    PubMed

    Devedjiev, Yancho D

    2015-02-01

    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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. Benchmarking of hospital information systems: Monitoring of discharge letters and scheduling can reveal heterogeneities and time trends

    PubMed Central

    Dugas, Martin; Eckholt, Markus; Bunzemeier, Holger

    2008-01-01

    Background Monitoring of hospital information system (HIS) usage can provide insights into best practices within a hospital and help to assess time trends. In terms of effort and cost of benchmarking, figures derived automatically from the routine HIS system are preferable to manual methods like surveys, in particular for repeated analysis. Methods Due to relevance for quality management and efficient resource utilization we focused on time-to-completion of discharge letters (assessed by CT-plots) and usage of patient scheduling. We analyzed these parameters monthly during one year at a major university hospital in Germany. Results We found several distinct patterns of discharge letter documentation indicating a large heterogeneity of HIS usage between different specialties (completeness 51 – 99%, delays 0 – 90 days). Overall usage of scheduling increased during the observation period by 62%, but again showed a considerable variation between departments. Conclusion Regular monitoring of HIS key figures can contribute to a continuous HIS improvement process. PMID:18423046

  1. 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.

  2. 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.

  3. Comprehensive alanine-scanning mutagenesis of Escherichia coli CsrA defines two subdomains of critical functional importance.

    PubMed

    Mercante, Jeffrey; Suzuki, Kazushi; Cheng, Xiaodong; Babitzke, Paul; Romeo, Tony

    2006-10-20

    The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA'-'lacZ, flhDC'-'lacZ, and pgaA'-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (beta1, residues 2-7) and containing the last (beta5, residues 40-47) beta-strands of CsrA. The beta1 and beta5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.

  4. 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.

  5. EMS mutagenesis in the pea aphid Acyrthosiphon pisum.

    PubMed

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

    2014-04-16

    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.

  6. Mitochondrial mutagenesis correlates with the local inflammatory environment in arthritis.

    PubMed

    Harty, Leonard C; Biniecka, Monika; O'Sullivan, Jacintha; Fox, Edward; Mulhall, Kevin; Veale, Douglas J; Fearon, Ursula

    2012-04-01

    To examine the association between mitochondrial mutagenesis and the proinflammatory microenvironment in patients with inflammatory arthritis. Fifty patients with inflammatory arthritis underwent arthroscopy and synovial tissue biopsies, synovial fluid and clinical assessment were obtained. Fifteen patients pre/post-TNFi therapy were also recruited. Normal synovial biopsies were obtained from 10 subjects undergoing interventional arthroscopy. Macroscopic synovitis/vascularity was measured by visual analogue scale. Cell-specific markers CD3 (T cells) and CD68 (macrophages) were quantified by immunohistology. TNFα, IL-6, IFNγ and IL-1β were measured in synovial fluids by MSD multiplex assays. Synovial tissue mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay (RMCA). The direct effect of TNFα on oxidative stress and mitochondrial function was assessed in primary cultures of rheumatoid arthritis synovial fibroblast cells (RASFCs). Mitochondrial mutagenesis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and mitochondrial mass (MM) were quantified using the RMCA and specific cell fluorescent probes. A significant increase in mtDNA mutation frequency was demonstrated in inflamed synovial tissue compared with control (p<0.05), an effect that was independent of age. mtDNA mutations positively correlated with macroscopic synovitis (r=0.52, p<0.016), vascularity (r=0.54, p<0.01) and with synovial fluid cytokine levels of TNFα (r=0.74, p<0.024) and IFNγ (r=0.72, p<0.039). mtDNA mutation frequency post-TNFi therapy was significantly lower in patients with a DAS<3.2 (p<0.05) and associated with clinical and microscopic measures of disease (p<0.05). In vitro TNFα significantly induced mtDNA mutations, ROS, MM and MMP in RASFCs (all p<0.05). High mitochondrial mutations are strongly associated with synovial inflammation showing a direct link between mitochondrial mutations and key proinflammatory pathways.

  7. 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

  8. [Analysis of Belamcanda chinensis with space flight mutagenesis by FTIR].

    PubMed

    Zhang, Hong-Mei; Guan, Ying; Shi, Jin-Shan

    2009-07-01

    The amorphous active components of the space mutagenesis Belamcanda chinensis and the ground group were measured, compared and analyzed. The purpose was to get a comprehensive understanding of the changes in quality of the 4th generation space mutagenesis Belamcanda chinensis, accumulate data for further studies, and try to establish the quality criterions of space mutagenesis Belamcanda chinensis. The result shows that the FTIR spectra of the space sample are almost the same as that of the ground one in terms of the main absorption peaks positions and shapes, but there are obvious differences in intensities. The intensity of the absorption peak at 1 642 and 1 318 cm(-1) of the space group was remarkably enhanced than the ground group, but at 1 541, 1 456, 1 417 and 1 051 cm(-1) it was decreased compared to the ground group. At the same time, the peak at 1 642 cm(-1) of the stretching vibration of C=O, the characteristic absorption of the keto, was remarkably enhanced. The peaks at 1 541 and 1 456 cm(-1) were assigned to C-C groups, the peak at 1 417 cm(-1) was due to the -CH2- groups, the peak at 1 318 cm(-1) was the characteristic absorption of calcium oxalate monohydrate, and the peak at 1 051 cm(-1) was assigned to C-O groups. It was shown that the relative content of flavone was increased distinctly. Space mutation breeding is conducive to breeding new varieties of highly active ingredients, it is also one of the ways to innovate germplasm resources of Chinese medicines efficiently. The effect of the space group is expected to be enhanced than the ground group, but it needs to be proved through further research.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. Phenotypic heterogeneity in a bacteriophage population only appears as stress-induced mutagenesis.

    PubMed

    Yosef, Ido; Edgar, Rotem; Qimron, Udi

    2016-11-01

    Stress-induced mutagenesis has been studied in cancer cells, yeast, bacteria, and archaea, but not in viruses. In a recent publication, we present a bacteriophage model showing an apparent stress-induced mutagenesis. We show that the stress does not drive the mutagenesis, but only selects the fittest mutants. The mechanism underlying the observed phenomenon is a phenotypic heterogeneity that resembles persistence of the viral population. The new findings, the background for the ongoing debate on stress-induced mutagenesis, and the phenotypic heterogeneity underlying a novel phage infection strategy are discussed in this short manuscript.

  18. Functional Consequence of Positive Selection Revealed through Rational Mutagenesis of Human Myeloperoxidase

    PubMed Central

    Loughran, Noeleen B.; Hinde, Sara; McCormick-Hill, Sally; Leidal, Kevin G.; Bloomberg, Sarah; Loughran, Sinéad T.; O’Connor, Brendan; Ó'Fágáin, Ciarán; Nauseef, William M.; O’Connell, Mary J.

    2012-01-01

    Myeloperoxidase (MPO) is a member of the mammalian heme peroxidase (MHP) multigene family. Whereas all MHPs oxidize specific halides to generate the corresponding hypohalous acid, MPO is unique in its capacity to oxidize chloride at physiologic pH to produce hypochlorous acid (HOCl), a potent microbicide that contributes to neutrophil-mediated host defense against infection. We have previously resolved the evolutionary relationships in this functionally diverse multigene family and predicted in silico that positive Darwinian selection played a major role in the observed functional diversities (Loughran NB, O'Connor B, O'Fagain C, O'Connell MJ. 2008. The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions. BMC Evol Biol. 8:101). In this work, we have replaced positively selected residues asparagine 496 (N496), tyrosine 500 (Y500), and leucine 504 (L504) with the amino acids present in the ancestral MHP and have examined the effects on the structure, biosynthesis, and activity of MPO. Analysis in silico predicted that N496F, Y500F, or L504T would perturb hydrogen bonding in the heme pocket of MPO and thus disrupt the structural integrity of the enzyme. Biosynthesis of the mutants stably expressed in human embryonic kidney 293 cells yielded apoproMPO, the heme-free, enzymatically inactive precursor of MPO, that failed to undergo normal maturation or proteolytic processing. As a consequence of the maturational arrest at the apoproMPO stage of development, cells expressing MPO with mutations N496F, Y500F, L504T, individually or in combination, lacked normal peroxidase or chlorinating activity. Taken together, our data provide further support for the in silico predictions of positive selection and highlight the correlation between positive selection and functional divergence. Our data demonstrate that directly probing the functional importance of positive selection can provide important insights into understanding protein evolution. PMID:22355012

  19. 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

  20. 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.

  1. 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

  2. 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.

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

    PubMed Central

    Hingston, Patricia A.; Piercey, Marta J.

    2015-01-01

    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/cm2 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. PMID:26025900

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

    PubMed Central

    Schölkopf, Bernhard; Logothetis, Nikos K.; Panzeri, Stefano

    2010-01-01

    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

  5. 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

  6. 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.

  7. [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.

  8. 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

  9. A mariner transposon vector adapted for mutagenesis in oral streptococci

    PubMed Central

    Nilsson, Martin; Christiansen, Natalia; Høiby, Niels; Twetman, Svante; Givskov, Michael; Tolker-Nielsen, Tim

    2014-01-01

    This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen. PMID:24753509

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  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. 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.

  18. The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

    PubMed Central

    Maisnier-Patin, Sophie; Roth, John R.

    2015-01-01

    Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

    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. 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. Copyright 2004 The National Academy of Sciencs of the USA

  9. Environmental mutagenesis during the end-Permian ecological crisis

    PubMed Central

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

    2004-01-01

    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. PMID:15282373

  10. 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

  11. 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

  12. Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU).

    PubMed

    Salinger, Andrew P; Justice, Monica J

    2008-04-01

    INTRODUCTIONThis protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 × 10(-3) in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed.

  13. Statistical methods for building random transposon mutagenesis libraries.

    PubMed

    Will, Oliver

    2008-01-01

    During the construction of random transposon mutagenesis libraries, four essential statistical issues arise: (1) Computing basic probability results for number of open reading frame knockouts. (2) Estimating the number of new open reading frames that will be knockouts in the next set of clones. (3) Estimating the number of essential open reading frames. (4) Computing the probability that an open reading frame is essential given the distribution of insertions. This chapter examines these issues and evaluates potential solutions using three different approaches: Efron and Thisted's estimator, Will and Jacobs's parametric bootstrap, and Blades and Broman's Gibbs sampler. In doing so, this chapter provides guidance for using the R statistical project to solve these problems.

  14. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    PubMed

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-10-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.

  15. 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.

  16. 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

  17. Mechanisms of uv mutagenesis in yeast and E. coli

    SciTech Connect

    Lawrence, C.; Christensen, R.; Christensen, J.R.; O'Brien, T.

    1983-01-01

    Experiments investigating ultraviolet light mutagenesis in either bakers' yeast, Saccharomyces cerevisiae, or E. coli have led to the following conclusions. First, cyclobutane pyrimidine dimers cause most mutations in both organisms; pyrimidine adducts, such as PyC, can account at best for only a small proportion. 86 percent of forward mutations induced at the E. coli lacI locus can be abolished by photoreactivation under conditions which do not alter the level of recA induction. About 75 percent of the forward mutations induced at the CAN1 locus of yeast could be removed by photoreactivation, a value that lies within the range observed previously for the reversion of CYC1 alleles (60 percent - 97 percent). Second, about 10 percent of the lacI forward mutations are untargeted, a smaller fraction than found previously for cycl-91 reversion in yeast. It is not yet clear whether the two species are really different in this respect, of whether the cycl-91 reversion site is a typical of the yeast genome at large. Third, analysis of reversion frequencies of 20 mutant alleles suggests that about 10 to 25 percent of all replication errors produced by mutagenic mechanisms in uv-irradiated yeast involve additions or deletions of base-pairs, indicating that error-prone repair does not just produce substitutions. Last, the REV1 locus in yeast is concerned with the induction of frameshift mutations at some, but not all, genetic sites, just as found previously for substitution mutations. The function of the REV3 gene is more widely, though not universally, required while the function of the RAD6 gene, like that of the recA locus in E. coli, appears to be necessary for all kinds of uv mutagenesis. E coli genes comparable to REV1 and REV3 have not yet been described; conversely, there does not yet appear to be a yeast equivalent of umuC.

  18. 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.

  19. 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.

  20. Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria

    PubMed Central

    Ilmjärv, Tanel; Naanuri, Eve; Kivisaar, Maia

    2017-01-01

    Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source. PMID:28777807

  1. 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

  2. The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

    PubMed

    Shor, Erika; Fox, Catherine A; Broach, James R

    2013-01-01

    Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.

  3. The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

    PubMed Central

    Shor, Erika; Fox, Catherine A.; Broach, James R.

    2013-01-01

    Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors. PMID:23935537

  4. 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.

  5. 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.

  6. Optimal information size in trial sequential analysis of time-to-event outcomes reveals potentially inconclusive results because of the risk of random error.

    PubMed

    Miladinovic, Branko; Mhaskar, Rahul; Hozo, Iztok; Kumar, Ambuj; Mahony, Helen; Djulbegovic, Benjamin

    2013-06-01

    The current approach for evaluating the risk of random error in meta-analyses (MAs) using trial sequential analysis (TSA) can accommodate binary and continuous data but not time-to-event data. We conducted a TSA for time-to-event outcomes and applied the method to determine the risk of random error in MAs for treatments of multiple myeloma. Literature search identified 11 systematic reviews consisting of 23 MAs. Of the 23 MAs, 13 had overall survival and 10 had progression-free survival as outcome; 48% (11 of 23) reported statistically significant treatment effects. We calculated the optimal a priori diversity-adjusted information size (APDIS) based on the relative risk reduction of 15% and 25%. We also calculated the optimal low-bias information size (LBIS) and low-bias diversity-adjusted information size (LBDIS). Overall, under APDIS15%, 48% (11 of 23) of MAs were false negative (FN) and 17% (4 of 23) of MAs were false positive. Under APDIS25%, 34% (8 of 23) of MAs were false negative and 4% (1 of 23) of MAs were false positive. LBIS identified 30% (7 of 23) as false negative MAs and 4% (1 of 23) as false positive MAs, whereas LBDIS identified 52% (12 of 23) as false negative MAs and 4% (1 of 23) as false positive MAs. The new method demonstrates the possibility of incorporating time-to-event outcomes into TSA and reveals that some MAs have potentially inconclusive results. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Essential genes in the infection model of Pseudomonas aeruginosa-PCR-based signature-tagged mutagenesis.

    PubMed

    Kukavica-Ibrulj, Irena; Levesque, Roger C

    2015-01-01

    PCR-based signature tagged mutagenesis is an "en masse" screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based STM applied to Pseudomonas aeruginosa permitted the identification of genes essential for in vivo maintenance by transposon insertion and negative selection in a mixed population of bacterial mutants. The innovative adaptations and refinement of the technology presented here with P. aeruginosa STM mutants selected in the rat model of chronic lung infection have given critical information about genes essential for causing a chronic infection and a wealth of information about biological processes in vivo. The additional use of competitive index analysis for measurement of the level of virulence in vivo, microarray-based screening of selected prioritized STM mutants coupled to metabolomics analysis can now be attempted systematically on a genomic scale. PCR-based STM and combined whole-genome methods can also be applied to any organism having selectable phenotypes for screening.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

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

    PubMed Central

    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. PMID:23917124

  20. 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.

  1. Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells.

    PubMed

    Ivics, Zoltán; Izsvák, Zsuzsanna; Medrano, Gerardo; Chapman, Karen M; Hamra, F Kent

    2011-09-08

    We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. CRISPR/Cas9-mediated targeted mutagenesis in grape

    PubMed Central

    Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. PMID:28542349

  8. CRISPR/Cas9-mediated targeted mutagenesis in grape.

    PubMed

    Nakajima, Ikuko; Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi; Endo, Masaki

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. [KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis.

    PubMed

    Suzuki, Genjiro; Weissman, Jonathan S; Tanaka, Motomasa

    2015-11-19

    Eukaryotic cells are targeted by pathogenic viruses and have developed cell defense mechanisms against viral infection. In yeast, the cellular extrachromosomal genetic element [KIL-d] alters killer activity of M double-stranded RNA killer virus and confers cell resistance against the killer virus. However, its underlying mechanism and the molecular nature of [KIL-d] are unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed that [KIL-d] selectively increases the rate of de novo mutation in the killer toxin gene of the viral genome, producing yeast harboring a defective mutant killer virus with a selective growth advantage over those with WT killer virus. These results suggest that a prion-like [KIL-d] element reprograms the viral replication machinery to induce mutagenesis and genomic inactivation via the long-hypothesized mechanism of "error catastrophe." The findings also support a role for prion-like protein aggregates in cellular defense and adaptation. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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

  15. 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.

  16. Evaluation of Glucose Dehydrogenase and Pyrroloquinoline Quinine (pqq) Mutagenesis that Renders Functional Inadequacies in Host Plants.

    PubMed

    Naveed, Muhammad; Sohail, Younas; Khalid, Nauman; Ahmed, Iftikhar; Mumtaz, Abdul Samad

    2015-08-01

    The rhizospheric zone abutting plant roots usually clutches a wealth of microbes. In the recent past, enormous genetic resources have been excavated with potential applications in host plant interaction and ancillary aspects. Two Pseudomonas strains were isolated and identified through 16S rRNA and rpoD sequence analyses as P. fluorescens QAU67 and P. putida QAU90. Initial biochemical characterization and their root-colonizing traits indicated their potential role in plant growth promotion. Such aerobic systems, involved in gluconic acid production and phosphate solubilization, essentially require the pyrroloquinoline quinine (PQQ)- dependent glucose dehydrogenase (GDH) in the genome. The PCR screening and amplification of GDH and PQQ and subsequent induction of mutagenesis characterized their possible role as antioxidants as well as in growth promotion, as probed in vitro in lettuce and in vivo in rice, bean, and tomato plants. The results showed significant differences (p < or = 0.05) in parameters of plant height, fresh weight, and dry weight, etc., deciphering a clear and in fact complementary role of GDH and PQQ in plant growth promotion. Our study not only provides direct evidence of the in vivo role of GDH and PQQ in host plants but also reveals their functional inadequacy in the event of mutation at either of these loci.

  17. 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.

  18. [KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis

    PubMed Central

    Suzuki, Genjiro; Weissman, Jonathan S.; Tanaka, Motomasa

    2017-01-01

    SUMMARY Eukaryotic cells are targeted by pathogenic viruses and have developed cell defense mechanisms against viral infection. In yeast, the cellular extrachromosomal genetic element [KIL-d] alters killer activity of M double–stranded RNA killer virus and confers cell resistance against the killer virus. However, its underlying mechanism and the molecular nature of [KIL-d] is unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed [KIL-d] selectively increases the rate of de novo mutation in the killer toxin gene of the viral genome, producing yeast harboring a defective mutant killer virus with a selective growth advantage over those with WT killer virus. These results suggest that a prion-like [KIL-d] element reprograms the viral replication machinery to induce mutagenesis and genomic inactivation via the long-hypothesized mechanism of “error catastrophe”. The findings also support a role for prion-like protein aggregates in cellular defense and adaptation. PMID:26590718

  19. 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.

  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. 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

  2. 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.

  3. Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis▿

    PubMed Central

    Vidales, Luz E.; Cárdenas, Lluvia C.; Robleto, Eduardo; Yasbin, Ronald E.; Pedraza-Reyes, Mario

    2009-01-01

    Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism. PMID:19011023

  4. 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

  5. Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii

    PubMed Central

    Ribeiro, Orquídea; Magalhães, Frederico; Aguiar, Tatiana Q; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2013-01-01

    To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities.   A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants. PMID:23644277

  6. Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis.

    PubMed

    Li, Zhuojian; Chen, Xiaojia; Guo, Shujun; Zhang, Huihua; Dong, Haojun; Wu, Guoyi; Hong, An; Gu, Jun

    2017-01-01

    We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in Pichia pastoris. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90 °C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p < 0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB.

    PubMed

    Ihssen, Julian; Kowarik, Michael; Wiesli, Luzia; Reiss, Renate; Wacker, Michael; Thöny-Meyer, Linda

    2012-09-24

    Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis

  8. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB

    PubMed Central

    2012-01-01

    Background Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. Results In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette

  9. 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%.

  10. Large scale integration of drug-target information reveals poly-pharmacological drug action mechanisms in tumor cell line growth inhibition assays

    PubMed Central

    Knight, Richard A.; Gostev, Mikhail; Ilisavskii, Sergei; Willis, Anne E.; Melino, Gerry; Antonov, Alexey V.

    2014-01-01

    Understanding therapeutic mechanisms of drug anticancer cytotoxicity represents a key challenge in preclinical testing. Here we have performed a meta-analysis of publicly available tumor cell line growth inhibition assays (~ 70 assays from 6 independent experimental groups covering ~ 500 000 molecules) with the primary goal of understanding molecular therapeutic mechanisms of cancer cytotoxicity. To implement this we have collected currently available information on protein targets for molecules that were tested in the assays. We used a statistical methodology to identify protein targets overrepresented among molecules exhibiting cancer cytotoxicity with the particular focus of identifying overrepresented patterns consisting of several proteins (i.e. proteins “A” and “B” and “C”). Our analysis demonstrates that targeting individual proteins can result in a significant increase (up to 50-fold) of the observed odds for a molecule to be an efficient inhibitor of tumour cell line growth. However, further insight into potential molecular mechanisms reveals a multi-target mode of action: targeting a pattern of several proteins drastically increases the observed odds (up to 500-fold) for a molecule to be tumour cytotoxic. In contrast, molecules targeting only one protein but not targeting an additional set of proteins tend to be nontoxic. Our findings support a poly-pharmacology drug discovery paradigm, demonstrating that anticancer cytotoxicity is a product, in most cases, of multi-target mode of drug action PMID:24553133

  11. 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

  12. Chromosomal aberrations in resident small mammals at a petrochemical waste dump site: a natural model for analysis of environmental mutagenesis. [Peromyscus leucopus; Sigmodon hispidus

    SciTech Connect

    McBee, K.

    1985-01-01

    Small mammals of two species (Peromyscus leucopus and Sigmodon hispidus) were trapped at a locality polluted with a complex mixture of petrochemical waste products, heavy metals, and PCB's, and from two matched, uncontaminated localities. Three cytogenetic techniques were employed to evaluate the use of these resident small mammals as indicators of environmental mutagenesis. Each technique also was assessed for its power of resolution in characterizing the action of environmental mutagens. Standard karyological analysis of flow cytometric analysis clearly indicated significant differences in chromosomal aberrancy between animals collected at the polluted site and the uncontaminated sites. Examination of flow DNA histograms of Peromyscus from the polluted site revealed broadened and flattened G/sub 1/ peaks and increases in CVs (coefficients of variation) for DNA content. CVs in animals from the polluted site consistently fell outside confidence limits set around values from animals collected at the uncontaminated site. These patterns are characteristically seen in laboratory animals challenged with powerful clastogens which suggests that individuals at the polluted site may be experiencing similar clastogenic events. This study demonstrates that small mammals are a feasible test model for evaluating environmental mutagenesis. Evaluation of different cytogenetic techniques suggests that a battery of several assays will provide the most accurate characterization of the action of environmental mutagenesis.

  13. [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.

  14. 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.

  15. [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.

  16. 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

  17. 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.

  18. 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.

  19. Arsenic toxicity, mutagenesis, and carcinogenesis--a health risk assessment and management approach.

    PubMed

    Tchounwou, Paul B; Centeno, Jose A; Patlolla, Anita K

    2004-01-01

    A comprehensive analysis of published data indicates that arsenic exposure induces cardiovascular diseases, developmental abnormalities, neurologic and neurobehavioral disorders, diabetes, hearing loss, hematologic disorders, and various types of cancer. Although exposure may occur via the dermal, and parenteral routes, the main pathways of exposure include ingestion, and inhalation. The severity of adverse health effects is related to the chemical form of arsenic, and is also time- and dose-dependent. Recent reports have pointed out that arsenic poisoning appears to be one of the major public health problems of pandemic nature. Acute and chronic exposure to arsenic has been reported in several countries of the world where a large proportion of drinking water (groundwater) is contaminated with high concentrations of arsenic. Research has also pointed significantly higher standardized mortality rates for cancers of the bladder, kidney, skin, liver, and colon in many areas of arsenic pollution. There is therefore a great need for developing a comprehensive health risk assessment (RA) concept that should be used by public health officials and environmental managers for an effective management of the health effects associated with arsenic exposure. With a special emphasis on arsenic toxicity, mutagenesis, and carcinogenesis, this paper is aimed at using the National Academy of Science's RA framework as a guide, for developing a RA paradigm for arsenic based on a comprehensive analysis of the currently available scientific information on its physical and chemical properties, production and use, fate and transport, toxicokinetics, systemic and carcinogenic health effects, regulatory and health guidelines, analytical guidelines and treatment technologies.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Structure-based design of combinatorial mutagenesis libraries

    PubMed Central

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

    2015-01-01

    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. PMID:25611189

  6. 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.

  7. 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

  8. 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.

  9. Trans-NIH neuroscience initiatives on mouse phenotyping and mutagenesis.

    PubMed

    Moldin, S O; Farmer, M E; Chin, H R; Battey, J F

    2001-08-01

    In the post-genomic era, the laboratory mouse will excel as a premier mammalian system to study normal and disordered biological processes, in part because of low cost, but largely because of the rich opportunities that exist for exploiting genetic tools and technologies in the mouse to systematically determine mammalian gene function. Many robust models of human disease may therefore be developed, and these in turn will provide critical clues to understanding gene function. The full potential of the mouse for understanding many of the neural and behavioral phenotypes of relevance to neuroscientists has yet to be realized. With the full anatomy of the mouse genome at hand, researchers for the first time will be able to move beyond traditional gene-by-gene approaches and take a global view of gene expression patterns crucial for neurobiological processes. In response to an action plan for mouse genomics developed on the basis of recommendations from the scientific community, seven institutes of the National Institutes of Health (NIH) initiated in 1999 a mouse genetics research program that specifically focused on neurobiology and complex behavior. The specific goals of these neuroscience initiatives are to develop high-throughput phenotyping assays and to initiate genome-wide mutagenesis projects to identify hundreds of mutant strains with heritable abnormalities of high relevance to neuroscientists. Assays and mutants generated in these efforts will be made widely available to the scientific community, and such resources will provide neuroscientists unprecedented opportunities to elucidate the molecular mechanisms of neural function and complex behavior. Such research tools ultimately will permit the manipulation and analysis of the mouse genome, as a means of gaining insight into the genetic bases of the mammalian nervous system and its complex disorders.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  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. 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.

  17. 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

  18. Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase.

    PubMed

    He, M; Varoglu, M; Sherman, D H

    2000-05-01

    A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. (3)H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

  19. 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.

  20. 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

  1. 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.

  2. Improvement of chitinase Pachi with nematicidal activities by random mutagenesis.

    PubMed

    Chen, Junpeng; An, Yangdongfang; Kumar, Ashok; Liu, Ziduo

    2017-03-01

    Chitinase, an enzyme that can degrade the main compositions of insect intestine and cuticle, has been used in the bio-control field. Our previous work has reported the chitinase Pachi with nematicidal activity (Caenorhabditis elegans). In the present study, to improve the chitinolytic and nematicidal activities of Pachi, a random mutant library was constructed by error-prone PCR and screened by bacteriophage T7-based high-throughput screening system. One mutant, PachiN35D was obtained from about 10, 000 clones. The kinetics analysis revealed that PachiN35D exhibited a 63% decrease in Km value against chitosan, a 2.1-fold enhancement in kcat/Km value and a 1.2-fold increase in specific activity over the wild-type Pachi. Moreover, the mortality analysis against Caenorhabditis elegans showed that the 50% lethal concentration (LC50) of PachiN35D is 309.6±1.1μg/ml and a 20% increase in nematicidal activity over the wild-type Pachi (with a LC50 value of 387.3±31.7μg/ml). The structure modeling and superimposition indicated that the substitution N35D reduced the distance between substrate and substrate-binding site Asp141, finally resulting in an increase in substrate affinity, catalytic efficiency and specific activity. These results provide useful information for the study of structure-function relationship of Pachi and lay a foundation for its potential applications in agro-biotechnology. Copyright © 2016. Published by Elsevier B.V.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    PubMed Central

    Bulina, Maria E; Chudakov, Dmitry M; Mudrik, Nikolay N; Lukyanov, Konstantin A

    2002-01-01

    Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. PMID:11972899

  12. 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

  13. Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

    PubMed

    Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

    2016-07-31

    Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

  14. 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). Copyright © 2015 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.

    PubMed

    Ma, Jun-Yan; Cao, Hai-Long; Tan, Hai-Dong; Hu, Xue-Jun; Liu, Wu-Jun; Du, Yu-Guang; Yin, Heng

    2016-01-01

    Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 μM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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.…

  5. 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.…

  6. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

    PubMed

    Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

    2015-07-01

    DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

    PubMed Central

    Galhardo, Rodrigo S.; Do, Robert; Yamada, Masami; Friedberg, Errol C.; Hastings, P. J.; Nohmi, Takehiko; Rosenberg, Susan M.

    2009-01-01

    Stress-induced mutagenesis is a collection of mechanis