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In Vitro Transcription

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Techniques in Molecular Biology

Abstract

There are several reasons for carrying out in vitro transcription reactions. First, to obtain fundamental information about the mechanism of RNA synthesis; second, to learn more about the control of gene expression at the transcriptional level; third, to carry out functional studies on recombinant DNA molecules; and, fourth, as a preparative technique to synthesise RNA molecules such as hybridisation probes or precursors to mature RNA. Different types of in vitro transcription system have been developed from a wide range of animals, plants and micro-organisms. These systems fall into three general categories: isolated chromatin or nuclei, soluble cell extracts, and purified cell components. The first of these is a eukaryotic system in which transcription is carried out by endogenous DNA and RNA polymerase. The second type of system, the soluble extract, is a partially purified cell homogenate which can be used to transcribe exogenous DNA. The third, and more rigidly defined system, involves the use of purified RNA polymerase, and possibly transcription factors, with exogenous DNA.

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Further Reading

  • Harnes, B.D. and Higgins, S.J. (eds) (1984) Transcription and Translation: A Practical Approach. (IRL Press, Oxford and Washington DC)

    Google Scholar 

  • Wu, R., Grossman, L. and Moldave, K. (eds) (1983) Recombinant DNA Part C. Methods in Enzymology, 101 (Academic Press, London)

    Google Scholar 

References

  1. Stryer, L. (1981) Biochemistry (Freeman, San Francisco)

    Google Scholar 

  2. Glass, R.E. (1982) Gene Function. E. coli and Its Heritable Elements. (Croom Helm, London)

    Google Scholar 

  3. Lewin, B. (1985) Genes II (Wiley, New York, Singapore)

    Google Scholar 

  4. Nevins, J.R. (1983) The Pathway of Eukaryotic mRNA Formation, Ann. Rev. Biochem., 52, 441–446

    Article  Google Scholar 

  5. Losick, R. and Chamberlin, M. (eds) (1976) RNA Polymerase. (Cold Spring Harbor, New York)

    Google Scholar 

  6. Jacob, S.T. (1983) Enzymes of Nucleic Acid Synthesis and Modification, Volume II: RNA Enzymes (CRC Press Inc., Boca Raton, Florida)

    Google Scholar 

  7. Chamberlin, M., Kingston, R., Gilman, M., Wiggs, J. and DeVera, A. (1983) Isolation of Bacterial and Bacteriophage RNA Polymerases and Their Use in Synthesis of RNA in vitro, in Methods in Enzymology, 101, pp. 540–567 (Academic Press, London)

    Google Scholar 

  8. Beebee, T.J.C. and Butterworth, P.H.W. (1981) Eukaryotic DNA-dependent RNA Polymerases: An Evaluation of Their Role in the Regulation of Gene Expression, in Kolodny, G.M. (ed.) Eukaryotic Gene Regulation, Volume II, pp. 1–56 (CRC Press Inc., Boca Raton, Florida)

    Google Scholar 

  9. Guilfoyle, T.J. (1983) DNA-dependent RNA Polymerases of Plants and Lower Eukaryotes, in Jacob, S.T. (ed.) Enzymes of Nucleic Acid Synthesis and Modification, Volume II: RNA Enzymes (CRC Press, Boca Raton, Florida)

    Google Scholar 

  10. Bennett, J. and Ellis, R.J. (1973) Solubilisation of Membrane-bound DNA-dependent RNA Polymerase of Pea Chloroplasts, Biochem. Soc. Trans. I., 892–894

    Google Scholar 

  11. Marzluff, W.F. and Huang, R.C.C. (1984) Transcription of RNA in Isolated Nuclei, in Hames, B.D. and Higgins, S.J. (eds) Transcription and Translation: A Practical Approach, pp. 89–129 (IRL Press, Oxford and Washington DC)

    Google Scholar 

  12. Carmichael, G.G. and McMaster, G.K. (1980) The Analysis of Nucleic Acids in Gels Using Glyoxal and Acridine Orange, in Methods in Enzymology, 65, pp. 380–381 (Academic Press, London)

    Google Scholar 

  13. Slater, R.J. (1983) The Extraction and Fractionation of RNA, in Walker, J.M. and Gaastra, W. (eds) Techniques in Molecular Biology, pp. 113–133 (Croom Helm, London/Macmillan, New York)

    Google Scholar 

  14. Grierson, D. (1982) Gel Electrophoresis of RNA, in Rickwood, D. and Hames, B.D. (eds) Gel Electrophoresis of Nucleic Acids: A Practical Approach, pp. 1–38 (IRL Press, Oxford and Washington DC)

    Google Scholar 

  15. Berk, A.J. and Sharp, P.A. (1977) Sizing and Mapping of Early Adenovirus mRNAs by Gel Electrophoresis of SI Endonuclease Digested Hybrids, Cell, 12, 721–821

    Article  Google Scholar 

  16. Favalaro, J., Treisman, R. and Kamen, R. (1980) Transcription Maps of Polyoma Virus-specific RNA: Analysis by Two-dimensional Nuclease S1 Gel Mapping, in Methods in Enzymology, 65, pp. 718–749 (Academic Press, London)

    Google Scholar 

  17. Gilmour, R.S. (1984) Transcription of Chromatin, in Hames, B.D. and Higgins, S.J. (eds) Transcription and Translation: A Practical Approach, pp. 131–151 (IRL Press, Oxford and Washington DC)

    Google Scholar 

  18. Zasloff, M. and Felsenfield, G. (1977) Use of Mercury-substituted Ribonucleoside Triphosphates Can Lead to Artefacts in the Analysis of in vitro Chromatin Transcripts, Biochem. Biophys. Res. Commun., 75, 598–603

    Article  Google Scholar 

  19. Brown, T.D.K., Pragnell, I.B. and Paul, J. (1980) Transcription of Friend Virus Proviral Sequences in Isolated Nuclei, Eur. J. Biochem., 104, 459–467

    Article  Google Scholar 

  20. Gurney, T. and Foster, D.N. (1977) Non-aqueous Isolation of Nuclei from Cultured Cells, Meth. Cell Biol., 16, 45–68

    Article  Google Scholar 

  21. Slater, R.J., Venis, M.A. and Grierson, D. (1978) Characterisation of RNA Synthesis by Nuclei Isolated from Zea mays, Planta, 144, 89–93

    Article  Google Scholar 

  22. Luthe, D.S. and Quatrano, R.S. (1980) Transcription in Isolated Wheat Nuclei, I and II, Plant Physiol., 65, 305–313

    Article  Google Scholar 

  23. McKnight, G.S. and Palmiter, R.D. (1979) Transcriptional Regulation of the Ovalbumin and Conalbumin Genes by Steroid Hormones in Chick Oviduct, J. Biol. Chem., 254, 9050–9058

    Google Scholar 

  24. Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M. and Darnell Jr., J.E. (1981) Transcriptional Control in the Production of Liver-specific mRNAs, Cell, 23, 731–739

    Article  Google Scholar 

  25. Friedman, R.L., Manly, S.P., McMahon, M., Kerr, I.M. and Stark, G.R. (1984) Transcriptional and Posttranscriptional Regulation of Interferon-induced Gene Expression in Human Cells, Cell, 38, 745–755

    Article  Google Scholar 

  26. Beach, L.R., Spencer, D., Randall, P.J. and Higgins, T.J.U. (1985) Transcriptional and Post-transcriptional Regulation of Storage Protein Gene Expression in Sulfur-deficient Pea Seeds, Nucleic Acids Res., 13, 999–1013

    Article  Google Scholar 

  27. Mosinger, E., Batschauer, A., Schäfer, E. and Apel, K. (1985) Phytochrome Control of in vitro Transcription of Specific Genes in Isolated Nuclei from Barley (Hordeum vulgare), Eur. J. Biochem., 147, 137–142

    Article  Google Scholar 

  28. Grummt, I. (1981) Specific Transcription of Mouse rDNA in a Cell-free System that Mimics Control in vivo, Proc. Natl. Acad. Sci. USA, 78, 727–731

    Article  Google Scholar 

  29. Wu, G-J. (1978) Adenovirus DNA-directed Transcription of 5.5S rRNA in vitro, Proc. Natl. Acad. Sci. USA, 75, 2175–2179

    Article  Google Scholar 

  30. Manley, J.L., Fire, A., Cano, A., Sharp, P.A. and Gefter, M.L. (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract, Proc. Natl. Acad. Sci. USA, 77, 3855–3859

    Article  Google Scholar 

  31. Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983) Accurate Transcription Initiation by RNA Polymerase II in a Soluble Extract from Isolated Mammalian Nuclei, Nucleic Acids Res., 11, 1475–1489

    Article  Google Scholar 

  32. Manley, J.L., Fire, A., Samuels, M. and Sharp, P.A. (1983) In vitro Transcription: Whole Cell Extract, in Methods in Enzymology, 101, 568–581 (Academic Press, London)

    Google Scholar 

  33. Dignam, J.D., Martin, P.L., Shastry, B.S. and Reader, R.G. (1983) Eukaryotic Gene Transcription with Purified Components, in Methods in Enzymology, 101, pp. 582–597 (Academic Press, London)

    Google Scholar 

  34. Melton, D.A., Krieg, P., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Efficient in vitro Synthesis of Biologically Active RNA and RNA Hybridisation Probes from Plasmids Containing a Bacteriophage SP6 Promoter, Nucleic Acids Res., 12, 7035–7056

    Article  Google Scholar 

  35. Chen, H.Z. and Zubay, G. (1983) Prokaryotic Coupled Transcription-Translation, in Methods in Enzymology, 101, pp. 674–689 (Academic Press, London)

    Google Scholar 

  36. Gold, L.M. and Schweiger, M. (1971) Synthesis of Bacteriophage-specific Enzymes Directed by DNA in vitro, in Methods in Enzymology, 20, pp. 537–542 (Academic Press, London)

    Google Scholar 

  37. Zubay, G. (1973) In vitro Synthesis of Protein in Microbial Systems, Ann. Rev. Genet., 1, 267–288

    Article  Google Scholar 

  38. Link, G. (1984) DNA Sequence Requirements for the Accurate Transcription of a Protein-coding Plastid Gene in a Plastid in vitro System from Mustard (Sinapis alba L.), EMBOJ.,3, 1697–1704

    Google Scholar 

  39. Orozco, E.M., Mullet, J.E. and Chua, N.H. (1985) An in vitro System for Accurate Transcription Initiation of Chloroplast Protein Genes, Nucleic Acids Res., 13, 1283–1302

    Article  Google Scholar 

  40. Walberg, M.W. and Clayton, D.A. (1983) In vitro Transcription of Human Mitochondrial DNA, J. Biol. Chem., 258, 1268–1275

    Google Scholar 

  41. Clayton, D.A. (1984) Transcription of the Mammalian Mitochondrial Genome, Ann. Rev. Biochem., 53, 573–594

    Article  Google Scholar 

  42. Leaver, C.J. and Gray, M.W. (1982) Mitochondrial Genome Organisation and Expression in Higher Plants, Ann. Rev. Plant Physiol., 33, 373–402

    Article  Google Scholar 

  43. Whitfield, P.R. and Bottomley, W. (1983) Organisation and Structure of Chloroplast Genes, Ann. Rev. Plant Physiol., 34, 279–310

    Article  Google Scholar 

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Authors
  1. Robert J. Slater
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Editors and Affiliations

  1. Division of Biological and Environmental Sciences, The Hatfield Polytechnic, PO Box 109, College Lane, Hatfield, Hertfordshire, AL10 9AB, England

    John M. Walker (Principal Lecturer in Biochemistry) (Principal Lecturer in Biochemistry)

  2. Department of Infectious Diseases, Section of Bacteriology, University of Utrecht, PO Box 80.171, 3508 TD, Utrecht, The Netherlands

    Wim Gaastra (Associate Professor of Microbiology) (Associate Professor of Microbiology)

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© 1987 John M. Walker and Wim Gaastra

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Slater, R.J. (1987). In Vitro Transcription. In: Walker, J.M., Gaastra, W. (eds) Techniques in Molecular Biology. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-9799-5_12

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  • DOI: https://doi.org/10.1007/978-1-4615-9799-5_12

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-0-7099-3673-2

  • Online ISBN: 978-1-4615-9799-5

  • eBook Packages: Springer Book Archive

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