Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1982 Oct 25;10(20):6353–6362. doi: 10.1093/nar/10.20.6353

Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes.

R Contreras, H Cheroutre, W Degrave, W Fiers
PMCID: PMC326924  PMID: 6294601

Abstract

A simple and efficient method for direct in vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts were translated in vivo in Xenopus laevis oocytes and in vitro in reticulocyte and in wheat germ cell-free protein synthesizing systems.

Full text

PDF
6355

Images in this article

6356Image
on p.6356
6357Image
on p.6357
6358Image
on p.6358
6359Image
on p.6359
6360Image
on p.6360

Selected References

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

  1. Contreras R., Cheroutre H., Fiers W. A simple apparatus for injection of nanoliter quantities into Xenopus laevis oocytes. Anal Biochem. 1981 May 1;113(1):185–187. doi: 10.1016/0003-2697(81)90063-4. [DOI] [PubMed] [Google Scholar]
  2. Contreras R., Khorana H. G. Proceedings: Controlled transcription of the synthetic gene for tyrosine transfer RNA. Arch Int Physiol Biochim. 1976 Feb;84(1):145–147. [PubMed] [Google Scholar]
  3. Contreras R., Volckaert G., Thys F., Van de Voorde A., Fiers W. Nucleotide sequence of the restriction fragment Hind F-Eco RI2 of SV40 DNA. Nucleic Acids Res. 1977 Apr;4(4):1001–1014. doi: 10.1093/nar/4.4.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Derynck R., Content J., DeClercq E., Volckaert G., Tavernier J., Devos R., Fiers W. Isolation and structure of a human fibroblast interferon gene. Nature. 1980 Jun 19;285(5766):542–547. doi: 10.1038/285542a0. [DOI] [PubMed] [Google Scholar]
  5. Derynck R., Content J., DeClercq E., Volckaert G., Tavernier J., Devos R., Fiers W. Isolation and structure of a human fibroblast interferon gene. Nature. 1980 Jun 19;285(5766):542–547. doi: 10.1038/285542a0. [DOI] [PubMed] [Google Scholar]
  6. Derynck R., Remaut E., Saman E., Stanssens P., De Clercq E., Content J., Fiers W. Expression of human fibroblast interferon gene in Escherichia coli. Nature. 1980 Sep 18;287(5779):193–197. doi: 10.1038/287193a0. [DOI] [PubMed] [Google Scholar]
  7. Downey K. M., Jurmark B. S., So A. G. Determination of nucleotide sequences at promoter regions by the use of dinucleotides. Biochemistry. 1971 Dec 21;10(26):4970–4975. doi: 10.1021/bi00802a021. [DOI] [PubMed] [Google Scholar]
  8. Downey K. M., So A. G. Studies on the kinetics of ribonucleic acid chain initiation and elongation. Biochemistry. 1970 Jun 9;9(12):2520–2525. doi: 10.1021/bi00814a019. [DOI] [PubMed] [Google Scholar]
  9. Fiers W., Contreras R., Haegemann G., Rogiers R., Van de Voorde A., Van Heuverswyn H., Van Herreweghe J., Volckaert G., Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature. 1978 May 11;273(5658):113–120. doi: 10.1038/273113a0. [DOI] [PubMed] [Google Scholar]
  10. Gheysen D., Iserentant D., Derom C., Fiers W. Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene. Gene. 1982 Jan;17(1):55–63. doi: 10.1016/0378-1119(82)90100-7. [DOI] [PubMed] [Google Scholar]
  11. Kleppe R., Khorana H. G. Studies on polynucleotides. CXIX. Transcription of short double-stranded deoxyribonucleic acids of defined nucleotide sequences. J Biol Chem. 1972 Oct 10;247(19):6149–6156. [PubMed] [Google Scholar]
  12. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  16. Roberts B. E., Paterson B. M. 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 U S A. 1973 Aug;70(8):2330–2334. doi: 10.1073/pnas.70.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smith M. M., Reeve A. E., Huang R. C. Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes. Cell. 1978 Oct;15(2):615–626. doi: 10.1016/0092-8674(78)90030-2. [DOI] [PubMed] [Google Scholar]
  19. Terao T., Dahlberg J. E., Khorana H. G. Studies on polynucleotides. CXX. On the transcription of a synthetic 29-unit long deoxyribopolynucleotide. J Biol Chem. 1972 Oct 10;247(19):6157–6166. [PubMed] [Google Scholar]
  20. Volckaert G., Jou W. M., Fiers W. Analysis of 32P-labeled bacteriophage MS2 RNA by a mini-fingerprinting procedure. Anal Biochem. 1976 May 7;72:433–446. doi: 10.1016/0003-2697(76)90551-0. [DOI] [PubMed] [Google Scholar]
  21. Wei C. M., Moss B. Methylation of newly synthesized viral messenger RNA by an enzyme in vaccinia virus. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3014–3018. doi: 10.1073/pnas.71.8.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wimmer E., Chang A. Y., Clark J. M., Jr, Reichmann M. E. Sequence studies of satellite tobacco necrosis virus RNA. Isolation and characterization of a 5'-terminal trinucleotide. J Mol Biol. 1968 Nov 28;38(1):59–73. doi: 10.1016/0022-2836(68)90128-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES