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An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates
Abstract
A simple method is described for converting a standard rabbit reticulocyte cell‐free extract (lysate) into an mRNA‐dependent protein synthesis system. The lysate is preincubated with CaCl 2 and micrococcal nuclease, and then excess ethyleneglycol‐bis(2‐aminoethylether)‐N,N′‐tetraacetic acid is added to chelate the Ca 2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full‐sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.
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Citations
Cites
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
LAEMMLI, U. K. (1970) · Nature · doi:10.1038/227680a0 · Cited by 249,961
Cited by
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease
Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., & Rutter, W. J. (1979) · Biochemistry · doi:10.1021/bi00591a005 · Cited by 21,725
Occludin: a novel integral membrane protein localizing at tight junctions.
Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S., & Tsukita, S. (1993) · The Journal of cell biology · doi:10.1083/jcb.123.6.1777 · Cited by 2,580
Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., & Harlow, E. (1988) · Nature · doi:10.1038/334124a0 · Cited by 1,403
Regulation of terminal differentiation of cultured human keratinocytes by vitamin A
Fuchs, E., & Green, H. (1981) · Cell · doi:10.1016/0092-8674(81)90169-0 · Cited by 750
Pelletier, J., & Sonenberg, N. (1985) · Cell · doi:10.1016/0092-8674(85)90200-4 · Cited by 610
Metadata
- Authors
- PELHAM, H. R. B., & JACKSON, R. J.
- Venue
- European Journal of Biochemistry
- Publisher
- Wiley
- Published
- 1976-08
- Volume
- 67
- Issue
- 1
- Pages
- 247-256
- ISSN
- 0014-2956 (ISSN-L)
- 1432-1033
- OpenAlex
- W1970403529
- Cited by
- 3,926
- References
- 1
- FWCI
- 68.02
- Type
- journal article
- Language
- en
- Open access
- closed
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- PELHAM, H. R. B., & JACKSON, R. J. (1976). An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates. European Journal of Biochemistry. https://doi.org/10.1111/j.1432-1033.1976.tb10656.x
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.
