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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 86)

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 (see Chapters 29, 30, and 32). There are advantages, however, of using wheat germ extracts:
  1. 1

    The in vtvo competition of mRNAs for translation IS more accurately represented, making the wheat germ system preferable for studymg 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 calcmm-activated nuclease. There IS, therefore, less dlsruption of the in vivo situation and contamination with calcium ions is less harmful The ldentlfication of all sizes of exogenous mRNA-directed translation products IS faclhtated 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 processmg may be achieved by the addition of mlcrosomal 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

Creatine Kinase Wheat Germ Translation Product Translation System Microsomal Membrane 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Louise Olliver
    • 1
  • Anne Grobler-Rabie
    • 1
  • Charles D. Boyd
  1. 1.University of Medicine and Dentistry of New BrunswickNew Brunswick

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