Abstract
One essential step for the molecular dissection of gene function is gene inactivation. In the yeast Saccharomyces cerevisiae, elaborate tools for gene disruption are available. Gene disruption cassettes carrying completely heterologous marker genes flanked by short DNA segments homologous to the corresponding regions left and right of the gene to be deleted result in highly efficient one-step gene disruption events yielding usually more than 50% of the clones carrying the correctly disrupted gene. Presence of loxP sites flanking the disruption marker gene allows Cre recombinase-mediated marker rescue so that the marker can be used to disrupt another gene.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rothstein, R. (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 194, 281–301.
Johnston, M., Riles, L., and Hegemann, J. H. (2002) Gene disruption. Methods Enzymol. 350, 290–315.
Winzeler, E. A., Shoemaker, D. D., Astromoff, A., et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906.
Giaever, G., Chu, A. M., Ni, L., et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391.
Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., and Boles, E. (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett. 464, 123–128.
Gldener, U., Heck, S., Fiedler, T., Beinhauer, J. D., and Hegemann, J. H. (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Actids Res. 24, 2519–2524.
Gueldener, U., Heinisch, J., Koehler, G. J., Voss, D., and Hegemann, J. H. (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. 30, e23.
Pluthero, F. G. (1993) Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res. 21, 4850–4851.
Gietz, R. D. and Woods, R. A. (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96.
Sherman, F. (2002) Getting started with yeast. Methods Enzymol. 350, 3–41.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Hegemann, J.H., Güldener, U., Köhler, G.J. (2006). Gene Disruption in the Budding Yeast Saccharomyces cerevisiae . In: Xiao, W. (eds) Yeast Protocol. Methods in Molecular Biology, vol 313. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-958-3:129
Download citation
DOI: https://doi.org/10.1385/1-59259-958-3:129
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-437-1
Online ISBN: 978-1-59259-958-5
eBook Packages: Springer Protocols