Skip to main content

Preparation of Splicing-Competent Nuclear Extracts from Adenovirus-lnfected Cells

  • Protocol

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 21))

Abstract

Adenovirus has contributed significantly to our current understanding of the organization and expression of genes in eukaryotic cells. The most startling discovery was probably the observation that most adenovirus mRNAs are encoded from discontinuous DNA segments. The split-gene and RNA-splicing concepts were rapidly extended to other systems and accepted as dogma within a few months after their initial discovery. It was clear right from the beginning that most adenovirus-transcription units encode multiple alternatively spliced mRNAs: Many are translated into proteins that have unique biological activities. It was also shown that the accumulation of alternatively spliced mRNA was temporally regulated during virus infection. The shift from the early to late pattern of mRNA accumulation was shown to be dependent on viral late-protein synthesis (1).

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Imperiale, M., Akusjarvi, G., and leppard, K. (1995) Post-transcriptional control of adenovirus gene expression. Curr Topics Microbiol 199, 139–171.

    Article  CAS  Google Scholar 

  2. Moore, M., Query, C. C., and Sharp, P. A. (1993) Splicing of precursors to mRNAs by the spliceosome, in the The RNA World (Gesteland, R. F., and Atkins, J. F., eds.)., Cold Spring Harbor Laboratory, Cold Spring Harbor,NY, 303–357.

    Google Scholar 

  3. Weingartner, B., and Keller, W. (1981) Transcription and processing of adenoviral RNA by extracts from HeLa cells Proc Natl. Acad Sci USA 78, 4092–4096

    Article  PubMed  CAS  Google Scholar 

  4. Kole, R., and Weissman, S. M. (1982) Accurate in vitro splicing of human β-globin RNA Nucleic Acids Res 10, 5429–5445.

    Article  PubMed  CAS  Google Scholar 

  5. Hernandez, N., and Keller, W. (1983) Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts Cell 35, 89–99.

    Article  PubMed  CAS  Google Scholar 

  6. Padgett, R. A., Hardy, S.F., and Sharp, P A. (1983) Splicing of adenovirus RNA in a cell-free transcription system Proc Natl Acad Sci USA 80, 5230–5234.

    Article  PubMed  CAS  Google Scholar 

  7. Kramer, A. R., Maniatis, T., Ruskm, 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

    Article  Google Scholar 

  8. Green, M. R., Maniatis, T., and Melton, D. A. (1983) Human β-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32, 681–694.

    Article  PubMed  CAS  Google Scholar 

  9. 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  PubMed  CAS  Google Scholar 

  10. Kreivi, J.-P., Zerivitz, K., and Akusjarvi, G., (1991) Sequences involved in the control of adenovirus L1 alternative RNA splicing. Nucleic Acids Res. 19, 2379–2386.

    Article  PubMed  CAS  Google Scholar 

  11. Zenvitz, K., Kreivi, J.-P., and Akusjarvi, G., (1992) Evidence for a HeLa cell splicing activity that is necessary for activation of a regualted adenovirus 3′ splice site. Nucleic Acids Res. 20, 3955–3961.

    Article  Google Scholar 

  12. Kreivi, J.-P., Zerivitz, K., and Akusjarvi, G., (1991) A U1 snRNA binding site improves the efficiency of in vitro pre-mRNA splicing. Nucleic Acids Res 19, 6956

    Article  PubMed  CAS  Google Scholar 

  13. Lee, K., Zerivitz, K., and Akusjarvi, G., (1995) Small-scale preparation of nuclear extracts from mammalian cells, in Cell Biology A Laboratory Handbook, vol 1 (Celis, J. E., ed.), Academic, London, pp. 668–673.

    Google Scholar 

  14. Eperon, I. C., and Kramer, A. R., (1993) Splicing of mRNA precursors in mammalian cells, in RNA Processing, A Practical Approach, vol 1 (Higgins, S. J. and Hames, B. D., eds.), IRL, Oxford, 757–101.

    Google Scholar 

  15. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J A, and Struhl, K. (1995) Preparation of nuclear and cytoplasmic extracts from mammalian cells, in Current Protocols in Molecular Biology. Wiley, New York, pp 12.1 1–12 1.9

    Google Scholar 

  16. Muhlemann, O., Kreivi, J.-P., and Akusjarvi, G (1995) Enhanced splicing of nonconsensus 3’ splice sites late during adenovirus infection J Virol. 69, 7324–7327.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Mühlemann, O., Akusjärvia, G. (1999). Preparation of Splicing-Competent Nuclear Extracts from Adenovirus-lnfected Cells. In: Wold, W.S.M. (eds) Adenovirus Methods and Protocols. Methods in Molecular Medicine™, vol 21. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-551-4:203

Download citation

  • DOI: https://doi.org/10.1385/0-89603-551-4:203

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-551-5

  • Online ISBN: 978-1-59259-603-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics