In Vitro Translation and Analysis of Early Events in Protein Synthesis Initiation in Nonreticulocyte Mammalian Cells
The most commonly used systems to translate mRNAs in vitro are the rabbit reticulocyte lysate and the wheat germ extract (1,2 and see Vol. 2 in this series). Both these systems have a number of advantages, including the ease and cheapness of preparation, the relative absence of RNAse, the high level of reinitiation of ribosomes onto mRNA, the high fidelity of translation, and the maintenance of activity upon long-term storage. Both of them have also been shown, upon addition of dog pancreatic membranes, to support processing of translated peptides (1). Furthermore, the reticulocyte has been the major source of information concerning the mechanism and control of protein synthesis in mammalian cells (2). For the investigation of many of the regulatory mechanisms in mammalian cells, however, neither the reticulocyte lysate nor the wheat germ system will be suitable. One, after all, is derived from plant cells and the other from a very specialized mammalian cell It is, therefore, unwise to assume that control mechanisms discovered in either of these are applicable to mammalian cells in general.
KeywordsVortex Glycerol Phenol Glycol Fractionation
- 1.Clemens, M. J. (1984) Translation of Eukaryotic Messenger RNA in Cell-Free Extracts, in Transcription and Translation: A Practical Approach (Hames, B.D. and Higgins, S. J., eds.) IRL Press, Oxford and Washington.Google Scholar
- 5.Austin, S. A., Pollard, J. W., Jagus, R., and Clemens, M. J. (1986) Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases. Eur. J. Biochem. 157, 39–47.PubMedCrossRefGoogle Scholar
- 7.Clemens, M. J., Galpine, A., Austin, S. A., Panniers, R., Henshaw, E. C., Duncan, R., Hershey, J. W. B., and Pollard, J. W. (1987) Regulation of polypeptide chain initiation in CHO cells with a temperature-sensitive leucyl-tRNA synthetase: Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor, GEF. J. Biol Chem. 262, 767–771.PubMedGoogle Scholar