In Vitro Translation of Messenger RNA in a Wheat Germ Extract Cell-Free System

  • Louise Olliver
  • Anne Grobler-Rabie
  • Charles D. Boyd
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 58)

Abstract

The wheat germ extract in vitro translation system has been used widely for faithful and efficient translation of viral and eukaryotic messenger RNAs in a heterologous cell-free system (1, 2, 3, 4, 5, 6, 7, 8, 9). With respect to the yield of translation products, the wheat germ extract is less efficient than most reticulocyte lysate cell-free systems. There are advantages, however, of using wheat germ extracts:
  1. 1.

    The in vivo competition of mRNAs for translation is more accurately represented, making the wheat germ system preferable for studying regulation of translation (1).

     
  2. 2.

    Particularly low levels of endogenous mRNA and the endogenous nuclease activity (10) obviate the requirement for treatment with a calcium-activated nuclease. There is, therefore, less disruption of the in vivo situation and contamination with calcium ions is less harmful. The identification of all sizes of exogenous mRNA-directed translation products is facilitated because of the low levels of endogenous mRNA present,

     
  3. 3.

    There is no posttranslational modification of translation products; primary products are therefore investigated, although processing may be achieved by the addition of microsomal membranes to the translation reaction.

     
  4. 4.

    The ionic conditions of the reaction may be altered to optimize the translation of large or small RNAs (2) (see Note 1).

     

Keywords

Magnesium Phenol Hydroxide Chloroform Electrophoresis 

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Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Louise Olliver
    • 1
  • Anne Grobler-Rabie
    • 1
  • Charles D. Boyd
    • 2
  1. 1.MRC Unit for Molecular and Cellular CardiologyUniversity of Stellenbosch Medical SchoolTygerbergSouth Africa
  2. 2.University of Medicine and Dentistry of New JerseyNew Brunswick

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