DOCSLIB.ORG

Isolation and Characterization of L-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Isolation and Characterization of L-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase UNIVERSITY OF HAWAII UBRAR,( Isolation and characterization of l-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and putrescine N-methyl transferase (PMT) complementary deoxyribonucleic acid (eDNA) in Nicotiana benthamiana using cytoplasmic inhibition of gene expression (CIGE) technology A THESIS SUBMIl lED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MOLECULAR BIOSCIENCES AND BIOENGINEERING AUGUST 2006 By J Malkeet Singh Thesis Committee: Monto Hiroshi Kumagai, Chairperson Dulal Bortbakur Winston Su We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Molecular Biosciences and Bioengineering. THESIS COMMlITEE ii ACKNOWLEDGEMENTS This research was funded by U.S. Department of Agriculture (USDA; TSTAR Gram: Patented research materials were kindly provided by Dr. Monto Kumagai and Dr. Guy della-Cioppa and Large Scale Biology Corpolation. Sincere thanks to all who have rendered encouragement and help: Dr. Monto Kumagai, Alain Ogura, Aaron Lani, Jessica Proberts, Naomi, Leah Tedder. Jennifer Busto, Rujunko Pugh, Manning Taite, Beth Irikura, Jason Dexter, Joanne Kurosawa, Anti Vesnefski, Dr. Dulal Bortbakur and Dr. Winston Suo Also to all family members and friends for their kindness and wisdom and especially to God who is sovereign over all. iii TABLE OF CONTENTS Aclmowledgements ..................................................................................iii List of Table ......................................................................................... vii . f· ... List 0 Figures .......................................................................................V111 Chapter 1. Literature Review .........................................................................1 1.1 Vual Vectors ..................................................................................... 1 1.1.1 The Use ofPlant Vual Vectors .............................................................. 1 1.1.2 Tobacco Mosaic VIrUS Vectors ............................................................. 3 1.1.3 Vual Vector Design Construct ............................................................ .4 1.2 Nicotiona benthamtana: A Model Plant for Functional Genomics ...................... 7 1.3 Safety and ContAinment Issues ................................................................ 8 1.4 Post Translational Gene Silencing (PTGS) ..................................................9 1.5 Metabolic Engineering ....................................................................... 13 1.6 Isoprenoid Biosynthesis and DXR. ........................................................... 14 1.7 Alkaloid biosynthesis and PMT ............................................................. .16 1.8 Aim ............................................................................................. 16 Chapter 2. Introduction .............................................................................. 18 2.1 DXR. and PMT ................................................................................. 18 2.1.1 DXR. in the MEP Pathway ................................................................ 18 2.1.2 PMT in the Alkaloid Pathway ............................................................ 20 iv Chapter 3. Materials and Methods ................................................................ 21 3.1 Bacterial Strains and Plants ..................................................................21 3.2 Recombinant VIral vector Assembly ...................................................... 21 3.3 eDNA Expression and Characterization ....................................................24 3.4 Plant RNA Isolation and Analysis ........................................................... 25 3.5 Microarray ....................................................................................... 26 3.5.1 Labeling and Hybridization for eDNA Microarrays ................................... 26 3.5.2 Microarray Scanning ....................................................................... 27 Chapter 4. Results ................................................................................... 28 4.1 DXR and PMT Cloning and Characterization .............................................28 4.1.1lsolation and Annotation ofN. benthamiana DXR. ................................... 28 4.1.2 DXR Sequeru:e Analysis .................................................................. 30 4.2 Construction of Recombinant VIral Vectors ...............................................33 4.3 RT PCR for the Verification ofInserts .....................................................35 4.4 Applications ofCIGE .........................................................................35 4.4.1 Mimicking Herbicide Effects inN. benthamiana ......................................35 4.4.2 Manipulation of Alkaloid Production in N. benthamiana .............................38 Chapter 5. Discussion ............................................................................... 39 5.1 Mimicking Herbicide Treatment ............................................................39 5.2 Manipulation of Biochemical Pathways ................................................... 40 5.3 Gene Silencing Systems ...................................................................... .43 v 5.4 DXRforDrugDesign ........................................................................ .45 5.4.1 Functioual Conservation across Plant, Protoman and Bacterial DXR ............ .45 5.4.2 Future Goals and Potential ofDXR Research ......................................... 47 5.5 References ...................................................................................... 50 vi USTOFTABLE 1 Genes in Putrescine related pathway ......................................................... 38 vii LIST OF FIGURES I The PTGS system ................................................................................ 12 2 Screening of eDNA libraries fur gene functions ............................................ 14 3 DXR in tile IPP biosynthesis pathway ...................................................................... 19 4 PMT in the nicotine biosynthesis pathway .................................................20 5 General flow chart of tile cloning of genes ................................................. 23 6 N. benthamiana DXR open reading frame .................................................. 29 7 Multiple alignment of plant, E.colt and P.jalcipan.nn meR.............................. 31 8 Recombinant viral vectors ..................................................................... 34 9 RTPCR........................................................................................... 35 10 Mimicking the effects of herbicides .......................................................... 37 11 OXR+. PMT- and GFP plants ............................................................... .42 12 Alignment between N. beothamiana OXR and E. coli DXR consensus sequence ........................................................................................ 46 13 N. benthamina (NBOXR) and P. falciparum (PFDXR) double alignment .............46 14 Molecular model ofthe conserved domain ofOXR...................................... .48 15 Flow chart of functional studies ...............................................................49 viii Chapter 1 Uterature Review 1.1 Viral Vectors 1.1.1 The Use of Plant Viral Vectors Transgenic crops have played a role in the production of pharmaceuticals and other valuable biological molecules. A more efficient strategy has involved by inoculating non-transgenic plants with virus-based vectors that carry foreign genes. As research progressed in the development of transgenic plants, it was discovered that plants have a built-in post-transcriptional gene silencing mechanism [8, 29]. The introduction of a gene homologous to an endogenous plant gene was found to cause a decrease in expression of both genes sucb as sbown in the case of controlling gene expression in transgenic petunia flowers [37]. This effect was also observed in response to homologous genes from viruses, implicating the gene silencing mechanism as a possible defense mechanism against viral genes [1]. With the development of infectious DNA clones, single-stranded RNA plant viruses have become key players in gene function discovery, metabolic engineering, and biomanufacturing. Viral expression vectors provide epigenetic expression of foreign sequences throughout infected plants, leading to gain-or loss of function phenotypes due to overexpression or cytoplasmic inhibition of gene expression. 1 Plant viruses are powerful transfection tools in molecular farming, producing pure, properly folded and gIycosylated proteins in plants faster and more economically than other expression systems [12, 13]. They are a highly desirable alternative to transgenic systems that require protracted periods to transform and regenerate whole plants, and that have variation in the expression levels of heterologous proteins. In transgenic systems, once a particular construct is inserted into the plant genome, it may take several crosses to establish a stable line in an elite cultivar. In contrast, plant viral vectors employed in the large-scale production of therapeutic drugs in greenhouse and field-grown crops directly yield high levels of foreign protein due to the rapid rate of viral
Load more

Recommended publications
  • Molecular Models for Shikimate Pathway Enzymes of Xylella Fastidiosa
    BBRC Biochemical and Biophysical Research Communications 320 (2004) 979–991 www.elsevier.com/locate/ybbrc Molecular models for shikimate pathway enzymes of Xylella fastidiosa Helen Andrade Arcuri,a,1 Fernanda Canduri,a,d,1 Jose Henrique Pereira,a Nelson Jose Freitas da Silveira,a Joao~ Carlos Camera Jr.,a Jaim Simoes~ de Oliveira,b Luiz Augusto Basso,b Mario Sergio Palma,c,d Diogenes Santiago Santos,e,* and Walter Filgueira de Azevedo Jr.a,d,* a Department of Physics IBILCE/UNESP, S~ao Jose do Rio Preto, SP 15054-000, Brazil b Rede Brasileira de Pesquisas em Tuberculose, Department of Molecular Biology and Biotecnology, UFRGS, Porto Alegre, RS 91501-970, Brazil c Laboratory of Structural Biology and Zoochemistry, Department of Biology, Institute of Biosciences, UNESP, Rio Claro, SP 13506-900, Brazil d Center for Applied Toxicology, Institute Butantan, S~ao Paulo, SP 05503-900, Brazil e Center for Research and Development in Molecular, Structural and Functional Molecular Biology, PUCRS 90619-900, Porto Alegre, RS, Brazil Received 25 May 2004 Available online 25 June 2004 Abstract The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants.
    [Show full text]
  • United States Patent (19) (11 Patent Number: 4,971,908 Kishore Et Al
    United States Patent (19) (11 Patent Number: 4,971,908 Kishore et al. 45 Date of Patent: Nov. 20, 1990 54 GLYPHOSATE-TOLERANT 56 References Cited 5-ENOLPYRUVYL-3-PHOSPHOSHKMATE U.S. PATENT DOCUMENTS SYNTHASE 4,769,061 9/1988 Comai ..................................... 71/86 (75. Inventors: Ganesh M. Kishore, Chesterfield; Primary Examiner-Robin Teskin Dilip M. Shah, Creve Coeur, both of Assistant Examiner-S. L. Nolan Mo. Attorney, Agent, or Firm-Dennis R. Hoerner, Jr.; 73 Assignee: Monsanto Company, St. Louis, Mo. Howard C. Stanley; Thomas P. McBride (21) Appl. No.: 179,245 57 ABSTRACT Glyphosate-tolerant 5-enolpyruvyl-3-phosphosikimate 22 Filed: Apr. 22, 1988 (EPSP) synthases, DNA encoding glyhphosate-tolerant EPSP synthases, plant genes encoding the glyphosate Related U.S. Application Data tolerant enzymes, plant transformation vectors contain ing the genes, transformed plant cells and differentiated 63 Continuation-in-part of Ser. No. 54,337, May 26, 1987, transformed plants containing the plant genes are dis abandoned. closed. The glyphosate-tolerant EPSP synthases are 51) Int. Cl. ....................... C12N 15/00; C12N 9/10; prepared by substituting an alanine residue for a glycine CO7H 21/04 residue in a conserved sequence found between posi 52 U.S. C. .............................. 435/172.1; 435/172.3; tions 80 and 120 in the mature wild-type EPSP syn 435/193; 536/27; 935/14 thase. 58) Field of Search............... 435/172.3, 193; 935/14, 935/67, 64 15 Claims, 14 Drawing Sheets U.S. Patent Nov. 20, 1990 Sheet 3 of 14 4,971,908 1. 50 Yeast . .TVYPFK DIPADQQKVV IPPGSKSSN RALITAATGE GQCKIKNLLH Aspergillus .
    [Show full text]
  • Lllllllllllllllllillllllllllllllilllllllllillllillllllllllll
    lllllllllllllllllIllllllllllllllIlllllllllIllllIllllllllllllllllllIllllllll U USOO53 10667A Umted States Patent [19] [11] Patent Number: 5,310,667 Eichholtz et a1. [45] Date of Patent: May 10, 1994 [54] GLYPHOSATE-TOLERANT 5-ENOLPYRUVYL-3-PHOSPHOSHIKIMATE OTHER PUBLICATIONS SYNTHASES Botterman et a1. (Aug. 1988) Trends in Genetics 4:219-222. [75] Inventors: David A_ Eichholtz, St Louis; Dassarma et al. (1986) Science 232:1242-1244. Charles S_ Gasser; Ganesh M_ Oxtoby et al. (1989) Euphytiza 40:173-180. Kishore, both of Chesterfield, an of Sezel, Enzyme Kineties, Behavior and Analysis of Rapid Mo_ Equilibrium and Steady State Enzyme System, John Wiley and Sons, New York, 1975, p. 15. [73] Assignee: Monsanto Company, St. Louis, Mo. Primary Examiner—Che S. Chereskiin Attorney, Agent, or Firm-Dennis R. Hoemer, Jr.; [21] Appl. No.: 380,963 RiChard H- Shear [57] ABSTRACT [22] Filed: Jul- 17’ 1989 Glyphosate-tolerant 5-enolpyruvy1-3-phosphoshikimate (EPSP) synthases, DNA encoding glyphosate-tolerant [51] Int. Cl.5 .................... .. C12N 15/01; C12N 15/29; EPSP synthases, plant genes encoding the glyphosate C12N 15/32 tolerant enzymes, plant transformation vectors contain [52] US. Cl. .............................. .. 435/ 172.3; 435/691; ing the genes, transformed plant cells and differentiated 800/205; 935/30; 935/35; 935/64; 536/236; transformed plants containing the plant genes are dis 536/23.7 closed. The glyphosate-tolerant EPSP synthases are [58] Field of Search ................... .. 435/68, 172.3, 69.1; prepared by substituting an alanine residue for a glycine 935/30, 35, 64, 67; 71/86, 113, 121; 530/370, residue in a ?rst conserved sequence found between 350; 800/205; 536/236, 23.7 positions 80 and 120, and either an aspartic acid residue ‘ or asparagine residue for a glycine residue in a second [56] References Cited conserved sequence found between positions 120 and 160 in the mature wild type EPSP synthase.
    [Show full text]
  • Supporting Information High-Throughput Virtual Screening
    Supporting Information High-Throughput Virtual Screening of Proteins using GRID Molecular Interaction Fields Simone Sciabola, Robert V. Stanton, James E. Mills, Maria M. Flocco, Massimo Baroni, Gabriele Cruciani, Francesca Perruccio and Jonathan S. Mason Contents Table S1 S2-S21 Figure S1 S22 * To whom correspondence should be addressed: Simone Sciabola, Pfizer Research Technology Center, Cambridge, 02139 MA, USA Phone: +1-617-551-3327; Fax: +1-617-551-3117; E-mail: [email protected] S1 Table S1. Description of the 990 proteins used as decoy for the Protein Virtual Screening analysis. PDB ID Protein family Molecule Res. (Å) 1n24 ISOMERASE (+)-BORNYL DIPHOSPHATE SYNTHASE 2.3 1g4h HYDROLASE 1,3,4,6-TETRACHLORO-1,4-CYCLOHEXADIENE HYDROLASE 1.8 1cel HYDROLASE(O-GLYCOSYL) 1,4-BETA-D-GLUCAN CELLOBIOHYDROLASE I 1.8 1vyf TRANSPORT PROTEIN 14 KDA FATTY ACID BINDING PROTEIN 1.85 1o9f PROTEIN-BINDING 14-3-3-LIKE PROTEIN C 2.7 1t1s OXIDOREDUCTASE 1-DEOXY-D-XYLULOSE 5-PHOSPHATE REDUCTOISOMERASE 2.4 1t1r OXIDOREDUCTASE 1-DEOXY-D-XYLULOSE 5-PHOSPHATE REDUCTOISOMERASE 2.3 1q0q OXIDOREDUCTASE 1-DEOXY-D-XYLULOSE 5-PHOSPHATE REDUCTOISOMERASE 1.9 1jcy LYASE 2-DEHYDRO-3-DEOXYPHOSPHOOCTONATE ALDOLASE 1.9 1fww LYASE 2-DEHYDRO-3-DEOXYPHOSPHOOCTONATE ALDOLASE 1.85 1uk7 HYDROLASE 2-HYDROXY-6-OXO-7-METHYLOCTA-2,4-DIENOATE 1.7 1v11 OXIDOREDUCTASE 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT 1.95 1x7w OXIDOREDUCTASE 2-OXOISOVALERATE DEHYDROGENASE ALPHA SUBUNIT 1.73 1d0l TRANSFERASE 35KD SOLUBLE LYTIC TRANSGLYCOSYLASE 1.97 2bt4 LYASE 3-DEHYDROQUINATE DEHYDRATASE
    [Show full text]
  • Architecture Génétique Des Caractères Cibles Pour La Culture Du Peuplier En Taillis À Courte Rotation Redouane El Malki
    Architecture génétique des caractères cibles pour la culture du peuplier en taillis à courte rotation Redouane El Malki To cite this version: Redouane El Malki. Architecture génétique des caractères cibles pour la culture du peuplier en taillis à courte rotation. Sciences agricoles. Université d’Orléans, 2013. Français. NNT : 2013ORLE2005. tel-00859626 HAL Id: tel-00859626 https://tel.archives-ouvertes.fr/tel-00859626 Submitted on 9 Sep 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DORLÉANS ÉCOLE DOCTORALE SANTE, SCIENCES BIOLOGIQUES ET CHIMIE DU VIVANT Unité de recherche Amélioration Génétique et Physiologie Forestières THÈSE présentée par : Redouane EL MALKI Soutenue le : 21 janvier 2013 pour obtenir le grade de : Docteur de luniversité dOrléans Discipline/ Spécialité : Biologie Architecture génétique des caractères cibles pour la culture du peuplier en taillis à courte rotation THÈSE dirigée par : Catherine BASTIEN Directrice de Recherche, INRA dOrléans RAPPORTEURS : Yves BARRIERE Directeur de Recherche, INRA de Lusignan Daniel
    [Show full text]
  • Translocation of the Precursor of 5-Enolpyruvylshikimate-3
    Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6873-6877, September 1986 Cell Biology Translocation of the precursor of 5-enolpyruvylshikimate-3- phosphate synthase into chloroplasts of higher plants in vitro (shikimate pathway/transit peptide/glyphosate) GUY DELLA-CIOPPA*, S. CHRISTOPHER BAUER, BARBARA K. KLEIN, DILIP M. SHAH, ROBERT T. FRALEY, AND GANESH M. KISHORE Monsanto Company, Plant Molecular Biology Group, Division of Biological Sciences, 700 Chesterfield Village Parkway, St. Louis, MO 63198 Communicated by Esmond E. Snell, June 16, 1986 ABSTRACT 5-enolPyruvylshikimate-3-phosphate syn- transferase; EC 2.5.1.19) catalyzes the transfer of the thase (EPSP synthase; 3-phosphoshikimate 1-carboxyvinyl- carboxyvinyl moiety of phosphoenolpyruvate (P-ePrv) to transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of shikimate-3-phosphate yielding EPSP and inorganic phos- the shikimate pathway in plants. This enzyme is the target for phate. EPSP synthase is an intermediate in the shikimate the nonselective herbicide glyphosate (N-phosphonomethyl- pathway that gives rise to the aromatic amino acids L- glycine). We have previously isolated a full-length cDNA clone phenylalanine, L-tyrosine, and L-tryptophan (8). In addition ofEPSP synthase from Petunia hybrida. DNA sequence analysis to the biosynthesis of these amino acids, the shikimate suggested that the enzyme is synthesized as a cytosolic precur- pathway is utilized for the production of p-amino- and sor (pre-EPSP synthase) with an amino-terminal transit, pep- p-hydroxybenzoic acids, and a variety of other natural plant tide. Based on the known amino terminus of the mature products (8). The biosynthesis of aromatic amino acids has enzyme, and the 5' open reading frame ofthe cDNA, the transit been shown to occur in isolated chloroplasts in vitro (9), and peptide of pre-EPSP synthase would be maximally 12 amino shikimate-pathway enzymes (including EPSP synthase) have acids long.
    [Show full text]
  • 8.2 Shikimic Acid Pathway
    CHAPTER 8 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORAromatic SALE OR DISTRIBUTION and NOT FOR SALE OR DISTRIBUTION Phenolic Compounds © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION CHAPTER OUTLINE Overview Synthesis and Properties of Polyketides 8.1 8.5 Synthesis of Chalcones © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 8.2 Shikimic Acid Pathway Synthesis of Flavanones and Derivatives NOT FOR SALE ORPhenylalanine DISTRIBUTION and Tyrosine Synthesis NOT FOR SALESynthesis OR DISTRIBUTION and Properties of Flavones Tryptophan Synthesis Synthesis and Properties of Anthocyanidins Synthesis and Properties of Isofl avonoids Phenylpropanoid Pathway 8.3 Examples of Other Plant Polyketide Synthases Synthesis of Trans-Cinnamic Acid Synthesis and Activity of Coumarins Lignin Synthesis Polymerization© Jonesof Monolignols & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Genetic EngineeringNOT FOR of Lignin SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Natural Products Derived from the 8.4 Phenylpropanoid Pathway Natural Products from Monolignols © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 119 © Jones & Bartlett Learning, LLC.
    [Show full text]
  • Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae C.-M
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1799-1804 Vol. 57, No. 6 0099-2240/91/061799-06$02.00/0 Copyright (C 1991, American Society for Microbiology Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae C.-M. LIU,* P. A. McLEAN, C. C. SOOKDEO, AND F. C. CANNONt BioTechnica International, Inc., 85 Bolton Street, Cambridge, Massachusetts 02140 Received 9 January 1991/Accepted 11 April 1991 Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). AR organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not as fast as on Pi. These results suggest that glyphosate degradation ability is widespread in the family Rhizobiaceae. Uptake and metabolism of glyphosate were studied by using R. meliloti 1021. Sarcosine was found to be the immediate breakdown product, indicating that the initial cleavage of glyphosate was at the C-P bond. Therefore, glyphosate breakdown in R. meliloti 1021 is achieved by a C-P lyase activity. Glyphosate (isopropylamine salt of N-phosphonomethyl- tained from Research Organics, Cleveland, Ohio. Agarose glycine) is the active ingredient in Roundup, a broad-spec- was obtained from International Biotechnology Inc., New trum postemergence herbicide sold worldwide for use in a Haven, Conn. large number of agricultural crops and industrial sites. It is a Culture of bacteria. Inocula of all rhizobia except Rhizo- potent inhibitor of the enzyme 3-enol-pyruvylshikimate-5- bium leguminosarum (strain 300) and ANU843 were grown phosphate synthase (EPSP synthase, EC 2.5.1.19), which is in LB (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% involved in the biosynthesis of the aromatic amino acids NaCl) at 28 to 32°C for 18 to 30 h.
    [Show full text]
  • Molecular and Phylogenetic Characterization of the Homoeologous EPSP Synthase Genes of Allohexaploid Wheat, Triticum Aestivum (L.) Attawan Aramrak1, Kimberlee K
    Aramrak et al. BMC Genomics (2015) 16:844 DOI 10.1186/s12864-015-2084-1 RESEARCH ARTICLE Open Access Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.) Attawan Aramrak1, Kimberlee K. Kidwell1, Camille M. Steber1,2*† and Ian C. Burke1*† Abstract Background: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized. Methods: Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring “Louise’. Development of genome- specific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis. Results: The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T.
    [Show full text]
  • Glyphosate's Suppression of Cytochrome P450 Enzymes
    Entropy 2013, 15, 1416-1463; doi:10.3390/e15041416 OPEN ACCESS entropy ISSN 1099-4300 www.mdpi.com/journal/entropy Review Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases Anthony Samsel 1 and Stephanie Seneff 2,* 1 Independent Scientist and Consultant, Deerfield, NH 03037, USA; E-Mail: [email protected] 2 Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, MA 02139, USA * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-617-253-0451; Fax: +1-617-258-8642. Received: 15 January 2013; in revised form: 10 April 2013 / Accepted: 10 April 2013 / Published: 18 April 2013 Abstract: Glyphosate, the active ingredient in Roundup®, is the most popular herbicide used worldwide. The industry asserts it is minimally toxic to humans, but here we argue otherwise. Residues are found in the main foods of the Western diet, comprised primarily of sugar, corn, soy and wheat. Glyphosate's inhibition of cytochrome P450 (CYP) enzymes is an overlooked component of its toxicity to mammals. CYP enzymes play crucial roles in biology, one of which is to detoxify xenobiotics. Thus, glyphosate enhances the damaging effects of other food borne chemical residues and environmental toxins. Negative impact on the body is insidious and manifests slowly over time as inflammation damages cellular systems throughout the body. Here, we show how interference with CYP enzymes acts synergistically with disruption of the biosynthesis of aromatic amino acids by gut bacteria, as well as impairment in serum sulfate transport. Consequences are most of the diseases and conditions associated with a Western diet, which include gastrointestinal disorders, obesity, diabetes, heart disease, depression, autism, infertility, cancer and Alzheimer’s disease.
    [Show full text]
  • Biochemical and Genetic Insights Into Asukamycin Biosynthesis Zhe Rui Louisiana State University and Agricultural and Mechanical College, [email protected]
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2011 Biochemical and genetic insights into asukamycin biosynthesis Zhe Rui Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Rui, Zhe, "Biochemical and genetic insights into asukamycin biosynthesis" (2011). LSU Doctoral Dissertations. 2028. https://digitalcommons.lsu.edu/gradschool_dissertations/2028 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. BIOCHEMICAL AND GENETIC INSIGHTS INTO ASUKAMYCIN BIOSYNTHESIS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Zhe Rui B.S., Zhejiang University, P. R. China, 2006 May 2011 ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my former major advisor Dr. Tin-Wein Yu, for his constant support and steadfast patience throughout my entire PhD study. His enthusiasm and meticulousness towards science have not only set a great example, but also guided me to become a devout believer in natural product engineering. My most enjoyable time in this research is the discussion session with him, where I have learned tremendously about logical thinking and scientific research development. In addition to a great mentor, he has been a true friend always available and ready to help.
    [Show full text]
  • 351 Section 2 General Information Concerning
    SECTION 2 GENERAL INFORMATION CONCERNING THE GENES AND THEIR ENZYMES THAT CONFER TOLERANCE TO GLYPHOSATE HERBICIDE Summary Note This document summarises the information available on the source of the genes that have been used to construct glyphosate-tolerant transgenic plants, the nature of the enzymes they encode, and the effects of the enzymes on the plant’s metabolism. Scope of this document: OECD Member countries agreed to limit this document to a discussion of the introduced genes and resulting enzymes that confer glyphosate tolerance to plants. The document is not intended to be an encyclopaedic review of all scientific experimentation with glyphosate-tolerant plants. In addition, this document does not discuss the wealth of information available on the herbicide glyphosate itself or the uses of the herbicide in agricultural and other applications. Food safety aspects of the use of glyphosate on glyphosate-tolerant transgenic plants are not discussed. Such information is available from other sources, including the respective governmental organisations which regulate the use of the herbicide. While the focus of this document is on the genes and enzymes involved in encoding glyphosate tolerance, reference is not made to specific plant species into which glyphosate tolerance might be introduced. Any issues relating to the cultivation of glyphosate-tolerant plants or to the potential for, or potential effects of, gene transfer from a glyphosate-tolerant plant to another crop plant or to a wild relative are outside the agreed scope of this document. It is intended, however, that this document should be used in conjunction with specific plant species biology Consensus Documents (see list of publications at the front of the document) when a biosafety assessment is made of plants with novel glyphosate herbicide resistance.
    [Show full text]