Summary
Temporal control of gene expression is a widespread feature of cell cycles, with clear transcriptional programs in bacteria, yeast, and metazoans. In budding yeast, approximately 1,000 genes are transcribed during a specific interval of the cell cycle. Although a number of factors that contribute to this periodic pattern of gene expression have been studied in Saccharomyces cerevisiae, pathways of cell cycle-regulated transcription remain largely undefined. To identify regulators of genes exhibiting cell cycle periodicity, we have developed a functional genomics approach termed reporter-based synthetic genetic array (R-SGA) analysis. Based on synthetic genetic array (SGA) analysis, R-SGA allows rapid and easily automated incorporation of a cell cycle reporter gene into the array of viable haploid yeast gene-deletion mutants. Scoring of reporter activity in mutant strains compared to wild type identifies candidate regulators of the cell cycle gene of interest. In contrast to microarrays, which generally provide information about the expression of all genes under a particular condition (for example, a single gene deletion), R-SGA analysis facilitates the study of the expression of a single gene in all deletion mutants. Our system can be adapted to examine the expression of any gene not only in the context of haploid deletion mutants but also using other array-based strain collections available to the yeast community.
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
Cho, R. J., Campbell, M. J., Winzeler, E. A., et al (1998). A genome-wide transcriptional analysis of the mitotic cell cycle. Mol. Cell. 2, 65–73.
Spellman, P. T., Sherlock, G., Zhang, M. Q., et al (1998). Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell. 9, 3273–3297.
Pramila, T., Wu, W., Miles, S., et al (2006). The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. Genes Dev. 20, 2266–2278.
Sopko, R., Huang, D., Preston, N., et al (2006). Mapping pathways and phenotypes by systematic gene overexpression. Mol. Cell. 21, 319–330.
Iyer, V. R., Horak, C. E., Scafe, C. S., et al (2001). Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533–538.
Simon, I., Barnett, J., Hannett, N., et al (2001). Serial regulation of transcriptional regulators in the yeast cell cycle. Cell 106, 697–708.
Horak, C. E., Luscombe, N. M., Qian, J., et al. (2002). Complex transcriptional circuitry at the G1/S transition in Saccharomyces cerevisiae. Genes Dev. 16, 3017–3033.
Lee, T. I., Rinaldi, N. J., Robert, F., et al (2002). Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804.
Ihmels, J., Friedlander, G., Bergmann, S., et al (2002). Revealing modular organization in the yeast transcriptional network. Nat. Genet. 31, 370–377.
Chen, H. C., Lee, H. C., Lin, T. Y., et al (2004). Quantitative characterization of the transcriptional regulatory network in the yeast cell cycle. Bioinformatics 20, 1914–1927.
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.
Tong, A. H., Evangelista, M., Parsons, A. B., et al (2001). Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368.
Giaever, G., Chu, A. M., Ni, L., et al (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391.
Tong, A. H., Boone, C. (2005). Synthetic genetic array analysis in Saccharomyces cerevisiae. In Yeast Protocols, 2nd edn., Methods in molecular biology, vol. 313 (Xiao, W., ed.).. Humana, Totowa, NJ, pp. 171–192.
Tong, A. H., Boone, C. (2007). High-throughput strain construction and systematic synthetic lethal screening in Saccharomyces cerevisiae. In Yeast Gene Analysis, 2nd edn., Methods in microbiology, vol. 36 (Stansfield, I. and Stark, M., eds.).. Elsevier, Burlington, MA, pp. 369–386; 706–707.
Johnson, A. D., Herskowitz, I. (1985). A repressor (MATα2 product). and its operator control expression of a set of cell type specific genes in yeast. Cell 42, 237–247.
Christianson, T. W., Sikorski, R. S., Dante, M., et al (1992). Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122.
Brachmann, C. B., Davies, A., Cost, G. J., et al (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.
Verma, R., Smiley, J., Andrews, B., Campbell, J. L. (1992). Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6. Proc. Natl. Acad. Sci. U S A. 89, 9479–9483.
Costanzo, M., Nishikawa, J. L., Tang, X., et al (2004). CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell 117, 899–913.
Cheng, T. H., Chang, C. R., Joy, P., et al (2000). Controlling gene expression in yeast by inducible site-specific recombination. Nucl. Acids Res. 28, E108.
Mnaimneh, S., Davierwala, A. P., Haynes, J., et al (2004). Exploration of essential gene functions via titratable promoter alleles. Cell 118, 31–44.
Acknowledgments
We thank Michael Costanzo and Jonathan Millman for sharing unpublished results. We are grateful to Helena Friesen for helpful discussions and critical reading of the manuscript. This work was supported by grants to B.A. from the Canadian Institutes for Health Research, the National Cancer Institute of Canada, and Genome Canada through the Ontario Genomics Institute.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sassi, H.E., Bastajian, N., Kainth, P., Andrews, B.J. (2009). Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating the Cell Cycle in Saccharomyces cerevisiae . In: Stagljar, I. (eds) Yeast Functional Genomics and Proteomics. Methods in Molecular Biology, vol 548. Humana Press. https://doi.org/10.1007/978-1-59745-540-4_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-540-4_4
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-934115-71-8
Online ISBN: 978-1-59745-540-4
eBook Packages: Springer Protocols