Document 0286
 DOCN  M94A0286
 TI    Expression of active, processed ricin in transgenic tobacco.
 DT    9412
 AU    Sehnke PC; Pedrosa L; Paul AL; Frankel AE; Ferl RJ; Department of
       Horticultural Sciences, University of Florida,; Gainesville 32611.
 SO    J Biol Chem. 1994 Sep 9;269(36):22473-6. Unique Identifier : AIDSLINE
       MED/94357881
 AB    The cDNA encoding the plant toxin precursor preproricin was introduced
       into tobacco via Agrobacterium tumefaciens-mediated gene transfer.
       Transgenic plants were assayed for type II ribosome-inactivating protein
       expression and activity. Western blot analysis of soluble leaf extracts
       using anti-ricin a-chain (RTA) antibodies identified 34- and 32-kDa
       proteins, which were electrophoretically indistinguishable from castor
       seed RTA. Analysis with anti-ricin b-chain (RTB) antibodies identified
       both a 34-kDa protein major band, which co-migrated with castor seed
       RTB, and a 30-kDa protein minor band. Enzyme-linked immunoassay of the
       transgenic leaf extracts with anti-RTA and anti-RTB indicated microgram
       per gram production on a fresh weight basis of soluble extractable
       recombinant ricin. Sugar binding enzyme-linked immunoassay employing an
       immobilized glycoprotein, asialofetuin, and anti-RTB antibodies
       confirmed the characteristic type II ribosome-inactivating protein
       galactose binding lectin activity of the recombinant ricin. The
       enzymatic activity of recombinant ricin was characterized for cell-free
       translation inhibition, as well as for overall cytotoxicity. A 50%
       inhibitory dose of 3 x 10(-11) M was observed for the immunoreactive
       leaf extract material using a rabbit reticulocyte translation inhibition
       assay, while a 50% lethal dose of 1 x 10(-12) M was calculated with
       human T-lymphotropic virus-1 infected leukemic T-cells.
 DE    Agrobacterium tumefaciens  Animal  Base Sequence  Castor
       Bean/GENETICS/METABOLISM  Cell Line  Cell Survival/DRUG EFFECTS  DNA
       Primers  DNA, Complementary/METABOLISM  Electrophoresis, Polyacrylamide
       Gel  Enzyme-Linked Immunosorbent Assay  *Gene Transfer  Human
       HTLV-I/DRUG EFFECTS/PHYSIOLOGY  Molecular Sequence Data  *Plants,
       Transgenic  Plasmids  Polymerase Chain Reaction  Protein
       Precursors/*BIOSYNTHESIS/ISOLATION & PURIF/TOXICITY  Rabbits
       Recombinant Proteins/BIOSYNTHESIS/ISOLATION & PURIF  Reticulocytes/DRUG
       EFFECTS  Ricin/*BIOSYNTHESIS/ISOLATION & PURIF/TOXICITY  Support, U.S.
       Gov't, P.H.S.  Tobacco/*METABOLISM  Translation, Genetic/DRUG EFFECTS
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).