Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1979 Apr;63(4):769–773. doi: 10.1104/pp.63.4.769

Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm 1

Linda Bowden-Bonnett a, J Michael Lord b
PMCID: PMC542914  PMID: 16660809

Abstract

Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3-day-old castor bean seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with molecular weights ranging from 10,000 to over 100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15 to 20% of the total translational products.

Attempts to reconstitute rough endoplasmic reticulum by the addition of washed castor bean microsomal membranes to the translational system were unsuccessful, these membranes severely inhibiting protein synthesis. Canine pancreatic microsomes could be added to endosperm messenger RNA-dependent reticulocyte lysates at relatively high concentrations while still allowing significant protein synthesis.

Full text

PDF
769

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BEEVERS H. Metabolic production of sucrose from fat. Nature. 1961 Jul 29;191:433–436. doi: 10.1038/191433a0. [DOI] [PubMed] [Google Scholar]
  2. Beevers H. Glyoxysomes of castor bean endosperm and their relation to gluconeogenesis. Ann N Y Acad Sci. 1969 Dec 19;168(2):313–324. doi: 10.1111/j.1749-6632.1969.tb43118.x. [DOI] [PubMed] [Google Scholar]
  3. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowden L., Lord J. M. Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm. Biochem J. 1976 Feb 15;154(2):491–499. doi: 10.1042/bj1540491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowden L., Lord J. M. The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm. Biochem J. 1976 Feb 15;154(2):501–506. doi: 10.1042/bj1540501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  8. Lord J. M., Bowden L. Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum. Plant Physiol. 1978 Feb;61(2):266–270. doi: 10.1104/pp.61.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Marcus A., Feeley J., Volcani T. Protein Synthesis in Imbibed Seeds III. Kinetics of Amino Acid Incorporation Ribosome Activation, and Polysome Formation. Plant Physiol. 1966 Sep;41(7):1167–1172. doi: 10.1104/pp.41.7.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  11. Scheele G., Dobberstein B., Blobel G. Transfer of proteins across membranes, Biosynthesis in vitro of pretrypsinogen and trypsinogen by cell fractions of canine pancreas. Eur J Biochem. 1978 Jan 16;82(2):593–599. doi: 10.1111/j.1432-1033.1978.tb12055.x. [DOI] [PubMed] [Google Scholar]
  12. Walk R. A., Hock B. Cell-free synthesis of glyoxysomal malate dehydrogenase. Biochem Biophys Res Commun. 1978 Mar 30;81(2):636–643. doi: 10.1016/0006-291x(78)91583-8. [DOI] [PubMed] [Google Scholar]
  13. Warren G., Dobberstein B. Protein transfer across microsomal membranes reassembled from separated membrane components. Nature. 1978 Jun 15;273(5663):569–571. doi: 10.1038/273569a0. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES