In Vitro Translation of mRNA in a Wheat Germ Extract Cell-Free System
互联网
606
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 ). |
