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Transcription In Vitro Using Bacteriophage RNA Polymerases

  • Elaine T. Schenborn

Abstract

Synthesis of specific RNA sequences in vitro is simplified because of the availability of bacteriophage RNA polymerases and specially designed DNA vectors. RNA polymerases encoded by SP6, T7, or T3 bacteriophage genomes recognize particular phage promoter sequences of their respective viral genes with a high degree of specificity (1, 2, 3). These RNA polymerases also transcribe DNA templates containing their cognate promoters under defined conditions in vitro (4,5). Standard reaction conditions for transcription in vitro can be adjusted for synthesis of large amounts of RNA or for smaller amounts of labeled RNA probes.

Keywords

Fresh Tube Isoamyl Alcohol Transcription Buffer Standard Reaction Condition Percent Incorporation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Butler, E. T. and Chamberlin, M. J. (1982) Bacteriophage SP6-specific RNA polymerase. J. Biol. Chem. 257, 5772–5778.PubMedGoogle Scholar
  2. 2.
    Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.PubMedCrossRefGoogle Scholar
  3. 3.
    Jorgensen, E. D., Joho, K., Risman, S., Moorefield, M. B., and McAllister, W. T. (1989) Promoter recognition by bacteriophage T3 and T7 RNA polymerases, in DNA-Protein Interaction in Transcription (Gralla, J. D., ed.), Liss, New York, pp. 79–88.Google Scholar
  4. 4.
    Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056.PubMedCrossRefGoogle Scholar
  5. 5.
    Krieg, P. A. and Melton, D. A. (1987) In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155, 397–415.PubMedCrossRefGoogle Scholar
  6. 6.
    Kaplan, G., Lubinski, J., Dasgupta, A., and Racaniello, V. R. (1985) In vitro synthesis of infectious poliovirus RNA. Proc. Natl Acad. Sci. USA 82, 8424–8248.PubMedCrossRefGoogle Scholar
  7. 7.
    Eggen, R., Verver, J., Wellink, J., DeJong, A., Goldbach, R., and van Kammen, A. (1989) Improvements of the infectivity of in vitro transcripts from cloned cowpea mosaic virus cDNA: impact of terminal nucleotide sequences. Virology 173, 447–455.PubMedCrossRefGoogle Scholar
  8. 8.
    Krainer, A. R., Maniatis, T., Ruskin, B., and Green, M. R. (1984) Normal and mutant human (β-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36, 993–1005.PubMedCrossRefGoogle Scholar
  9. 9.
    Krieg, P. A. and Melton, D. A. (1984) Formation of the 3′ end of histone mRNA by posttranscriptional processing. Nature 308, 203–206.PubMedCrossRefGoogle Scholar
  10. 10.
    Georgiev, O., Mous, J., and Birnstiel, M. (1984) Processing and nucleo-cytoplasmic transport of histone gene transcripts. Nucleic Acids Res. 12, 8539–8551.PubMedCrossRefGoogle Scholar
  11. 11.
    Krieg, P. A. and Melton, D. A. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12, 7057–7070.PubMedCrossRefGoogle Scholar
  12. 12.
    Burgin, A. B. and Pace, N. R. (1990) Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. EMBO J. 9, 4111–4118.PubMedGoogle Scholar
  13. 13.
    Heus, H. A., Uhlenbeck, O. C, and Pardi, A. (1990) Sequence-dependent structural variations of hammerhead RNA enzymes. Nucleic Acids Res. 18, 1103–1108.PubMedCrossRefGoogle Scholar
  14. 14.
    Nicole, L. M. and Tanguay, R. M. (1987) On the specificity of antisense RNA to arrest in vitro translation of mRNA coding for Drosophila hsp 23. Biosci. Rep. 7, 239–246.PubMedCrossRefGoogle Scholar
  15. 15.
    Melton, D. A. (1985) Injected antisense RNAs specifically block messenger RNA translation in vivo. Proc. Natl. Acad. Sci. USA 82, 144–148.PubMedCrossRefGoogle Scholar
  16. 16.
    Witherell, G. W., Wu, H.-N., and Uhlenbeck, O. C. (1990) Cooperative binding of R17 coat protein to RNA. Biochemistry 29, 11,051–11,057.PubMedCrossRefGoogle Scholar
  17. 17.
    Turek, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510.CrossRefGoogle Scholar
  18. 18.
    Langer, P. R., Waldrop, A. A., and Ward, D. C. (1982) Enzymatic synthesis of biotinlabeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633–6637.CrossRefGoogle Scholar
  19. 19.
    Aigner, S. and Pette, D. (1990) In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA. Histochemistry 95, 11–18.PubMedCrossRefGoogle Scholar
  20. 20.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  21. 21.
    Casey, J. and Davidson, N. (1977) Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 4, 1539–1552.PubMedCrossRefGoogle Scholar
  22. 22.
    Uhlig, H., Saeger, W., Fehr, S., and Ludecke, D. K. (1991) Detection of growth hormone, prolactin and human beta-chorionic gonadotropin messenger RNA in growth-hormone-secreting pituitary adenomas by in situ hybridization. Virchows Arch. Pathol. Anat. Histopathol. 418, 539–546.CrossRefGoogle Scholar
  23. 23.
    Matthaei, K. I. and Reed, K. C. (1986) Chromosome assignment in somatic hybrids by in situ hybridization with tritium labeled Riboprobe® RNA probes. Promega Notes 5, 5–6.Google Scholar
  24. 24.
    Zinn, K., DiMaio, D., and Maniatis, T. (1983) Identification of two distinct regulatory regions adjacent to the human (β-interferon gene. Cell 34, 865–879.PubMedCrossRefGoogle Scholar
  25. 25.
    Contreras, R., Cheroutre, H., Degrave, W., and Fiers, W. (1982) Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic Acids Res. 10, 6353–6362.PubMedCrossRefGoogle Scholar
  26. 26.
    Schenborn, E. T. and Mierendorf, R. C. (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 13, 6223–6236.PubMedCrossRefGoogle Scholar
  27. 27.
    Forster, A. C., Mclnnes, J. L., Skingle, D. C., and Symons, R. H. (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 13, 745–761.PubMedCrossRefGoogle Scholar
  28. 28.
    Gurevich, V. V., Pokrovskaya, I. D., Obukhova, T. A., and Zozulya, S. A. (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Analyt. Biochem. 195, 207–213.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Elaine T. Schenborn
    • 1
  1. 1.Promega CorporationMadison

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