In Vitro Translation of Messenger RNA 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 29, 30, and 32). There are advantages, however, of using wheat germ extracts:

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