An efficient mRNA-dependent translation system from reticulocyte lysates - PubMed (original) (raw)
An efficient mRNA-dependent translation system from reticulocyte lysates
H R Pelham et al. Eur J Biochem. 1976.
Free article
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 CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N'-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have neglibible 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 of 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 200000, 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.
Similar articles
- Excess of micrococcal nuclease may harm the exogenous mRNA in the mRNA-dependent rabbit reticulocyte cell-free system.
Neeleman L, van Vloten-Doting L. Neeleman L, et al. FEBS Lett. 1978 Nov 1;95(1):103-6. doi: 10.1016/0014-5793(78)80061-1. FEBS Lett. 1978. PMID: 720587 No abstract available. - Poly(A) dependent translation in rabbit reticulocyte lysate.
Wakiyama M, Futami T, Miura K. Wakiyama M, et al. Biochimie. 1997 Dec;79(12):781-5. doi: 10.1016/s0300-9084(97)86937-4. Biochimie. 1997. PMID: 9523021 - Coupled transcription-translation in chloroplast lysates.
Bard JD, Bourque DP, Zaitlin D. Bard JD, et al. Methods Enzymol. 1986;118:270-95. doi: 10.1016/0076-6879(86)18078-5. Methods Enzymol. 1986. PMID: 2419735 Review. No abstract available. - Multiple keratins of cultured human epidermal cells are translated from different mRNA molecules.
Fuchs E, Green H. Fuchs E, et al. Cell. 1979 Jul;17(3):573-82. doi: 10.1016/0092-8674(79)90265-4. Cell. 1979. PMID: 383305 Review. No abstract available.
Cited by
- Analysis of in vitro and in vivo products of the TMV 30kDa open reading frame using antisera raised against a synthetic peptide.
Kiberstis PA, Pessi A, Atherton E, Jackson R, Hunter T, Zimmern D. Kiberstis PA, et al. FEBS Lett. 1983 Dec 12;164(2):355-360. doi: 10.1016/0014-5793(83)80316-0. FEBS Lett. 1983. PMID: 33785967 Free PMC article. - Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes.
Walter P, Jackson RC, Marcus MM, Lingappa VR, Blobel G. Walter P, et al. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1795-9. doi: 10.1073/pnas.76.4.1795. Proc Natl Acad Sci U S A. 1979. PMID: 109833 Free PMC article. - Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucei.
Lheureux M, Lheureux M, Vervoort T, Van Meirvenne N, Steinert M. Lheureux M, et al. Nucleic Acids Res. 1979 Oct 10;7(3):595-609. doi: 10.1093/nar/7.3.595. Nucleic Acids Res. 1979. PMID: 116191 Free PMC article. - Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.
Bulleid NJ, Freedman RB. Bulleid NJ, et al. EMBO J. 1990 Nov;9(11):3527-32. doi: 10.1002/j.1460-2075.1990.tb07561.x. EMBO J. 1990. PMID: 2120042 Free PMC article. - Characterization of Rous sarcoma virus src gene products synthesized in vitro.
Beemon K, Hunter T. Beemon K, et al. J Virol. 1978 Nov;28(2):551-66. doi: 10.1128/JVI.28.2.551-566.1978. J Virol. 1978. PMID: 214578 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous