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

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 (1–9). With respect to the yield of translation products, the wheat germ extract is less efficient than most reticulocyte lysate cell-free systems (see Chapters 106, 107, and 109). There are advantages, however, of using wheat germ extracts:

• 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. Particularly low levels of endogenous mRNA and the endogenous nuclease activity (10) obviate the requirement for treatment with a calcium-activated nuclease. There is, there fore, 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. 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. The ionic conditions of the reaction may be altered to optimize the translation of large or small RNAs (2) (see Note 1).