SpringerProtocols: Abstract: In Vitro Translation of Messenger RNA in a Rabbit Reticulocyte Lysate Cell-Free System

In Vitro Translation of Messenger RNA in a Rabbit Reticulocyte Lysate Cell-Free System

By: C. L. Oliver¹, C. D. Boyd¹

Abstract

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The identification of specific messenger RNA molecules and the characterization of the proteins encoded by them, has been greatly assisted by the development of in vitro translation systems. These cell-free extracts comprise the cellular components necessary for protein synthesis, i.e., ribosomes, tRNA, rRNA, amino acids, initiation, elongation and termination factors, and the energy-generating system (1). Heterologous mRNAs are faithfully and efficiently translated in extracts of HeLa cells (2), Krebs II ascites tumor cells (2), mouse L cells (2), rat and mouse liver cells (3), Chinese hamster ovary (CHO) cells (2), and rabbit reticulocyte lysates (2,4), in addition to those of rye embryo (5) and wheat germ (6). Translation in cell-free systems is simpler and more rapid (60 min vs 24 h) than the in vivo translation system using Xenopus oocytes.

Affiliation(s): (1) MRC Unit for Molecular and Cellular Cardiology, University of Stellenbosch Medical School, Tygerberg, South Africa

Book Title: Nucleic Acids

Series: Methods in Molecular Biology | Volume: 2 | Pub. Date: Dec-11-1984 | Page Range: 145-155 | DOI: 10.1385/0-89603-064-4:145

Subject: Biochemistry

References

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1.	Lodish, H. F. (1976) Translational control of protein synthesis. Ann. Rev. Biochem. 45, 39–72.

2.	McDowell, M. J., Joklik, W. K., Villa-Komaroff, L., and Lodish, H. F. (1972) Translation of reovirus messenger RNAs synthesized in vitro into reovirus polypeptides by several mammalian cell-free extracts. Proc. Natl. Acad. Sci. USA 69, 2649–2653.

3.	Sampson, J., Mathews, M. B., Osborn, M, and Borghetti, A. F. (1972) Hemoglobin messenger ribonucleic acid translation in cell-free systems from rat and mouse liver and Landschutz ascites cells. Biochem. 11, 3636–3640.

4.	Pelham, H. R. B., and Jackson, R. J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247–256.

5.	Carlier, A. R., and Peumans, W. J. (1976) The rye embryo system as an alternative to the wheat-system for protein synthesis in vitro. Biochem. Biophys. Acta 447, 436–444.

6.	Roberts, B. E., and Paterson, B. M. (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc. Natl. Acad. Sci. USA 70, 2330–2334.

7.	Kessler, S. W. (1981) Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. In Methods in Enzymology (Langone, J J, and Van Vunakis, H., eds.) 73, 441–459. Academic Press, New York.

8.	Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

9.	Bonner, W. M., and Laskey, R. A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83–88.

10.	Shields, D., and Blobel, G. (1978) Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J. Biol. Chem. 253, 3753–3756.

11.	Jackson, R. C., and Blobel, G. (1977) Post-translational cleavage of presecretory proteins with an extract of rough microsomes, from dog pancreas, with signal peptidase activity. Proc. Natl. Acad. Sci. USA 74, 5598–5602.

12.	Karr, S. R., Rich, C. B., Foster, J. A., and Przybyla, A. (1981) Optimum conditions for cell-free synthesis of elastin. Coll. Res. 1, 73–81.

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