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. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057

Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.

P A Krieg, D A Melton
PMCID: PMC320142  PMID: 6207484

Abstract

We describe a method for the synthesis of microgram quantities of eucaryotic messenger RNAs. Injection into the cytoplasm of frog oocytes and addition to wheat germ extracts show that these synthetic RNAs function efficiently as messenger RNAs. We confirm that a 5' cap on the mRNA is essential for translation in injected oocytes and show that most of the 3' flanking region, including the poly A tail, can be deleted without the abolition of protein synthesis. The method of mRNA synthesis involves in vitro transcription of cDNAs which have been cloned into SP6 vectors (described in the accompanying paper). This method enables one to produce large amounts of mRNA and consequently protein from any cDNA clone.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allende C. C., Allende J. E., Firtel R. A. The degradation of ribonucleic acids injected into Xenopus laevis oocytes. Cell. 1974 Jul;2(3):189–196. doi: 10.1016/0092-8674(74)90093-2. [DOI] [PubMed] [Google Scholar]
  2. Butler E. T., Chamberlin M. J. Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. J Biol Chem. 1982 May 25;257(10):5772–5778. [PubMed] [Google Scholar]
  3. Cavalieri R. L., Havell E. A., Vilcek J., Pestka S. Synthesis of human interferon by Xenopus laevis oocytes: two structural genes for interferons in human cells. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3287–3291. doi: 10.1073/pnas.74.8.3287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colman A., Morser J. Export of proteins from oocytes of Xenopus laevis. Cell. 1979 Jul;17(3):517–526. doi: 10.1016/0092-8674(79)90260-5. [DOI] [PubMed] [Google Scholar]
  5. Darnell J. E., Jr Transcription units for mRNA production in eukaryotic cells and their DNA viruses. Prog Nucleic Acid Res Mol Biol. 1979;22:327–353. doi: 10.1016/s0079-6603(08)60803-x. [DOI] [PubMed] [Google Scholar]
  6. Goldberg D. A. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5794–5798. doi: 10.1073/pnas.77.10.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  8. Gurdon J. B. Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J Embryol Exp Morphol. 1976 Dec;36(3):523–540. [PubMed] [Google Scholar]
  9. Gurdon J. B., Lane C. D., Woodland H. R., Marbaix G. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature. 1971 Sep 17;233(5316):177–182. doi: 10.1038/233177a0. [DOI] [PubMed] [Google Scholar]
  10. Gurdon J. B., Lingrel J. B., Marbaix G. Message stability in injected frog oocytes: long life of mammalian alpha and beta globin messages. J Mol Biol. 1973 Nov 5;80(3):539–551. doi: 10.1016/0022-2836(73)90421-x. [DOI] [PubMed] [Google Scholar]
  11. Huez G., Marbaix G., Hubert E., Cleuter Y., Leclercq M., Chantrenne H., Devos R., Soreq H., Nudel U., Littauer U. Z. Readenylation of polyadenylate-free globin messenger RNA restores its stability in vivo. Eur J Biochem. 1975 Nov 15;59(2):589–592. doi: 10.1111/j.1432-1033.1975.tb02486.x. [DOI] [PubMed] [Google Scholar]
  12. Huez G., Marbaix G., Hubert E., Leclercq M., Nudel U., Soreq H., Salomon R., Lebleu B., Revel M., Littauer U. Z. Role of the polyadenylate segment in the translation of globin messenger RNA in Xenopus oocytes. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3143–3146. doi: 10.1073/pnas.71.8.3143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  14. Krieg P. A., Melton D. A. Formation of the 3' end of histone mRNA by post-transcriptional processing. Nature. 1984 Mar 8;308(5955):203–206. doi: 10.1038/308203a0. [DOI] [PubMed] [Google Scholar]
  15. Kronenberg M. N., Roberts B. E., Efstratiadis A. The 3' noncoding region of beta-globin mRNA is not essential for in vitro translation. Nucleic Acids Res. 1979 Jan;6(1):153–166. doi: 10.1093/nar/6.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  18. Lenk R., Ransom L., Kaufmann Y., Penman S. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell. 1977 Jan;10(1):67–78. doi: 10.1016/0092-8674(77)90141-6. [DOI] [PubMed] [Google Scholar]
  19. Lockard R. E., Lane C. Requirement for 7-methylguanosine in translation of globin mRNA in vivo. Nucleic Acids Res. 1978 Sep;5(9):3237–3247. doi: 10.1093/nar/5.9.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Martin S. A., Paoletti E., Moss B. Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions. J Biol Chem. 1975 Dec 25;250(24):9322–9329. [PubMed] [Google Scholar]
  21. Monroy G., Spencer E., Hurwitz J. Purification of mRNA guanylyltransferase from vaccinia virions. J Biol Chem. 1978 Jun 25;253(12):4481–4489. [PubMed] [Google Scholar]
  22. Paterson B. M., Rosenberg M. Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature. 1979 Jun 21;279(5715):692–696. doi: 10.1038/279692a0. [DOI] [PubMed] [Google Scholar]
  23. Patient R. K., Harris R., Walmsley M. E., Williams J. G. The complete nucleotide sequence of the major adult beta globin gene of Xenopus laevis. J Biol Chem. 1983 Jul 25;258(14):8521–8523. [PubMed] [Google Scholar]
  24. Perry R. P. Processing of RNA. Annu Rev Biochem. 1976;45:605–629. doi: 10.1146/annurev.bi.45.070176.003133. [DOI] [PubMed] [Google Scholar]
  25. Richter J. D., Smith L. D. Differential capacity for translation and lack of competition between mRNAs that segregate to free and membrane-bound polysomes. Cell. 1981 Nov;27(1 Pt 2):183–191. doi: 10.1016/0092-8674(81)90372-x. [DOI] [PubMed] [Google Scholar]
  26. Richter J. D., Wasserman W. J., Smith L. D. The mechanism for increased protein synthesis during Xenopus oocyte maturation. Dev Biol. 1982 Jan;89(1):159–167. doi: 10.1016/0012-1606(82)90304-9. [DOI] [PubMed] [Google Scholar]
  27. Roberts B. E., Gorecki M., Mulligan R. C., Danna K. J., Rozenblatt S., Rich A. Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system. Proc Natl Acad Sci U S A. 1975 May;72(5):1922–1926. doi: 10.1073/pnas.72.5.1922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  29. Taniguchi T., Mantei N., Schwarzstein M., Nagata S., Muramatsu M., Weissmann C. Human leukocyte and fibroblast interferons are structurally related. Nature. 1980 Jun 19;285(5766):547–549. doi: 10.1038/285547a0. [DOI] [PubMed] [Google Scholar]
  30. Williams J. G., Kay R. M., Patient R. K. The nucleotide sequence of the major beta-globin mRNA from Xenopus laevis. Nucleic Acids Res. 1980 Sep 25;8(18):4247–4258. doi: 10.1093/nar/8.18.4247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]

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