Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique

Nature Protocols volume 1, pages 2517–2526 (2006)Cite this article

This article has been updated

Abstract

In the heterokaryon rescue technique, gene deletions are carried out using the pyrG nutritional marker to replace the coding region of target genes via homologous recombination in Aspergillus nidulans. If an essential gene is deleted, the null allele is maintained in spontaneously generated heterokaryons that consist of two genetically distinct types of nuclei. One nuclear type has the essential gene deleted but has a functional pyrG allele (pyrG+). The other has the wild-type allele of the essential gene but lacks a functional pyrG allele (pyrG−). Thus, a simple growth test applied to the uninucleate asexual spores formed from primary transformants can identify deletions of genes that are non-essential from those that are essential and can only be propagated by heterokaryon rescue. The growth tests also enable the phenotype of the null allele to be defined. Diagnostic PCR can be used to confirm deletions at the molecular level. This technique is suitable for large-scale gene-deletion programs and can be completed within 3 weeks.

Note: In the version of this article initially published, the black ball in Figure 2c was incorrectly described as representing a pyrG–, geneX+ nuclei. This ball represents pyrG+, geneX–. The error has been corrected in all versions of the article.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Example of anticipated results when a non-essential gene is deleted.
Figure 2: Outline of heterokaryon rescue.19
Figure 3: Example of anticipated results when an essential gene is deleted.
Figure 4: Examples of the cell types used in the heterokaryon rescue protocol.

Similar content being viewed by others

Change history

  • 25 January 2007

    In the version of this article initially published, the black ball in Figure 2c was incorrectly described as representing a pyrG–, geneX+ nuclei. This ball represents pyrG+, geneX–. The error has been corrected in all versions of the article.

References

  1. Pontecorvo, G., Roper, J.A., Hemmons, L.M., MacDonald, K.D. & Bufton, A.W. Advances in Genetics (ed. Demerec, M.) 141–238 (Academic Press, New York, 1953).

    Google Scholar 

  2. Osmani, S.A., Engle, D.B., Doonan, J.H. & Morris, N.R. Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. Cell 52, 241–251 (1988).

    Article  CAS  Google Scholar 

  3. O'Donnell, K.L., Osmani, A.H., Osmani, S.A. & Morris, N.R. bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans. J. Cell Sci. 99, 711–719 (1991).

    CAS  PubMed  Google Scholar 

  4. Martin, M.A., Osmani, S.A. & Oakley, B.R. The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans. J. Cell Sci. 110, 623–633 (1997).

    CAS  PubMed  Google Scholar 

  5. Prigozhina, N.L. et al. Gamma-tubulin plays an essential role in the coordination of mitotic events. Mol. Biol. Cell 15, 1374–1386 (2004).

    Article  CAS  Google Scholar 

  6. Osmani, A.H., Davies, J., Liu, H.L., Nile, A. & Osmani, S.A. Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans. Mol. Biol. Cell 17, 4946–4961 (2006).

    Article  CAS  Google Scholar 

  7. Chaveroche, M.K., Ghigo, J.M. & d'Enfert, C. A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 28, E97 (2000).

    Article  CAS  Google Scholar 

  8. Yang, L. et al. Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans. Eukaryot. Cell 3, 1359–1362 (2004).

    Article  CAS  Google Scholar 

  9. Szewczyk, E. et al. Fusion PCR and gene targeting in Aspergillus nidulans. Nature Protocols DOI: 10.1038/nprot.2006.405 (2006).

  10. Peters, J., King, R.W., Hoog, C. & Kirschner, M.W. Identification of BIME as a subunit of the anaphase-promoting complex. Science 274, 1199–1201 (1996).

    Article  CAS  Google Scholar 

  11. Nayak, T. et al. A versatile and efficient gene targeting system for Aspergillus nidulans. Genetics 172, 1557–1566 (2006).

    Article  CAS  Google Scholar 

  12. Jung, M.K., Prigozhina, N., Oakley, C.E., Nogales, E. & Oakley, B.R. Alanine-scanning mutagenesis of Aspergillus gamma-tubulin yields diverse and novel phenotypes. Mol. Biol. Cell 12, 2119–2136 (2001).

    Article  CAS  Google Scholar 

  13. Lu, K.P., Rasmussen, C.D., May, G.S. & Means, A.R. Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans. Mol. Endocrinol. 6, 365–374 (1992).

    CAS  PubMed  Google Scholar 

  14. McGoldrick, C.A., Gruver, C. & May, G.S. myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth. J. Cell Biol. 128, 577–587 (1995).

    Article  CAS  Google Scholar 

  15. Som, T. & Kolaparthi, V. Developmental decisions in Aspergillus nidulans are modulated by ras activity. Mol. Cell. Biol. 14, 5333–5348 (1994).

    Article  CAS  Google Scholar 

  16. Bussink, H.J. & Osmani, S.A. A cyclin-dependent kinase family member (PHOA) is required to link developmental fate to environmental conditions in Aspergillus nidulans. EMBO J. 17, 3990–4003 (1998).

    Article  CAS  Google Scholar 

  17. Ye, X.S., Fincher, R.R., Tang, A., Osmani, A.H. & Osmani, S.A. Regulation of the anaphase-promoting complex/cyclosome by BIMAAPC3 and proteolysis of NIMA. Mol. Biol. Cell 9, 3019–3030 (1998).

    Article  CAS  Google Scholar 

  18. Oakley, B.R. & Osmani, S.A. in The Cell Cycle, a Practical Approach (eds. Fantes, P. & Brooks, R.) 127–142 (Oxford University Press, New York, 1993).

    Google Scholar 

  19. In the version of this article initially published, the black ball in Figure 2c was incorrectly described as representing a pyrG–, geneX+ nuclei. This ball represents pyrG+, geneX–. The error has been corrected in all versions of the article.

Download references

Acknowledgements

This work was supported by grant GM042564 to S.A.O. and grant GM31837 to B.R.O. from the US National Institutes of Health. We thank members of the Osmani lab for reading the manuscript and members of our labs for their contributions to the development of the protocols described in this work.

Author information

Authors and Affiliations

  1. Department of Molecular Genetics, The Ohio State University, Columbus, 43210, Ohio, USA

    Aysha H Osmani, Berl R Oakley & Stephen A Osmani

Authors
  1. Aysha H Osmani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Berl R Oakley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephen A Osmani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stephen A Osmani.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Osmani, A., Oakley, B. & Osmani, S. Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique. Nat Protoc 1, 2517–2526 (2006). https://doi.org/10.1038/nprot.2006.406

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.406

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing