Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter (original) (raw)

Abstract

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

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Selected References

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  1. Angerer L. M., Angerer R. C. Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res. 1981 Jun 25;9(12):2819–2840. doi: 10.1093/nar/9.12.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  3. Birchmeier C., Schümperli D., Sconzo G., Birnstiel M. L. 3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1057–1061. doi: 10.1073/pnas.81.4.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blattner F. R., Dahlberg J. E. RNA synthesis startpoints in bacteriophage lambda: are the promoter and operator transcribed? Nat New Biol. 1972 Jun 21;237(77):227–232. doi: 10.1038/newbio237227a0. [DOI] [PubMed] [Google Scholar]
  5. Brahic M., Haase A. T. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6125–6129. doi: 10.1073/pnas.75.12.6125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cox K. H., DeLeon D. V., Angerer L. M., Angerer R. C. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol. 1984 Feb;101(2):485–502. doi: 10.1016/0012-1606(84)90162-3. [DOI] [PubMed] [Google Scholar]
  10. Diaz M. O., Barsacchi-Pilone G., Mahon K. A., Gall J. G. Transcripts from both strands of a satellite DNA occur on lampbrush chromosome loops of the newt Notophthalmus. Cell. 1981 Jun;24(3):649–659. doi: 10.1016/0092-8674(81)90091-x. [DOI] [PubMed] [Google Scholar]
  11. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Green M. R., Roeder R. G. Definition of a novel promoter for the major adenovirus-associated virus mRNA. Cell. 1980 Nov;22(1 Pt 1):231–242. doi: 10.1016/0092-8674(80)90171-3. [DOI] [PubMed] [Google Scholar]
  15. Guerrier-Takada C., Altman S. Catalytic activity of an RNA molecule prepared by transcription in vitro. Science. 1984 Jan 20;223(4633):285–286. doi: 10.1126/science.6199841. [DOI] [PubMed] [Google Scholar]
  16. Hernandez N., Keller W. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell. 1983 Nov;35(1):89–99. doi: 10.1016/0092-8674(83)90211-8. [DOI] [PubMed] [Google Scholar]
  17. Kassavetis G. A., Butler E. T., Roulland D., Chamberlin M. J. Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription. J Biol Chem. 1982 May 25;257(10):5779–5788. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Kruger K., Grabowski P. J., Zaug A. J., Sands J., Gottschling D. E., Cech T. R. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982 Nov;31(1):147–157. doi: 10.1016/0092-8674(82)90414-7. [DOI] [PubMed] [Google Scholar]
  21. Lau P. P., Gray H. B., Jr Extracellular nucleases of Alteromonas espejiana BAL 31.IV. The single strand-specific deoxyriboendonuclease activity as a probe for regions of altered secondary structure in negatively and positively supercoiled closed circular DNA. Nucleic Acids Res. 1979 Jan;6(1):331–357. doi: 10.1093/nar/6.1.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Manley J. L. Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate. Cell. 1983 Jun;33(2):595–605. doi: 10.1016/0092-8674(83)90440-3. [DOI] [PubMed] [Google Scholar]
  23. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maxwell I. H., Maxwell F., Hahn W. E. Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2' (3')-phosphate. Nucleic Acids Res. 1977 Jan;4(1):241–246. doi: 10.1093/nar/4.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McAllister W. T., Morris C., Rosenberg A. H., Studier F. W. Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J Mol Biol. 1981 Dec 15;153(3):527–544. doi: 10.1016/0022-2836(81)90406-x. [DOI] [PubMed] [Google Scholar]
  27. Meinkoth J., Wahl G. Hybridization of nucleic acids immobilized on solid supports. Anal Biochem. 1984 May 1;138(2):267–284. doi: 10.1016/0003-2697(84)90808-x. [DOI] [PubMed] [Google Scholar]
  28. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  29. Miele E. A., Mills D. R., Kramer F. R. Autocatalytic replication of a recombinant RNA. J Mol Biol. 1983 Dec 15;171(3):281–295. doi: 10.1016/0022-2836(83)90094-3. [DOI] [PubMed] [Google Scholar]
  30. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  31. Padgett R. A., Hardy S. F., Sharp P. A. Splicing of adenovirus RNA in a cell-free transcription system. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5230–5234. doi: 10.1073/pnas.80.17.5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Peebles C. L., Ogden R. C., Knapp G., Abelson J. Splicing of yeast tRNA precursors: a two-stage reaction. Cell. 1979 Sep;18(1):27–35. doi: 10.1016/0092-8674(79)90350-7. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Roberts J. W. Termination factor for RNA synthesis. Nature. 1969 Dec 20;224(5225):1168–1174. doi: 10.1038/2241168a0. [DOI] [PubMed] [Google Scholar]
  36. Rosenberg M., Weissman S., deCrombrugghe B. Termination of transcription in bacteriophage lambda. Heterogeneous, 3'-terminal oligo-adenylate additions and the effects of rho factor. J Biol Chem. 1975 Jun 25;250(12):4755–4764. [PubMed] [Google Scholar]
  37. Weaver R. F., Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zinn K., DiMaio D., Maniatis T. Identification of two distinct regulatory regions adjacent to the human beta-interferon gene. Cell. 1983 Oct;34(3):865–879. doi: 10.1016/0092-8674(83)90544-5. [DOI] [PubMed] [Google Scholar]