A Method for Gene Disruption That Allows Repeated Use of USR3 Selection in the Construction of Multiply Disrupted Yeast Strains

Eric Alani ¹, Liang Cao ¹, and Nancy Kleckner ¹

¹ Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138

In this paper, we describe a 3.8-kb molecular construct that we have used to disrupt yeast genes. The construct consists of a functional yeast URA3 gene flanked by 1.1-kb direct repeats of a bacterial sequence. It is straightforward to insert the 3.8-kb segment into a cloned target gene of interest and then introduce the resulting disruption into the yeast genome by integrative transformation. An appropriate DNA fragment containing the disruption plus flanking homology can be obtained by restriction enzyme digestion. After introducing such fragments into yeast by transformation, stable integrants can be isolated by selection for Ura⁺. The important feature of this construct that makes it especially useful is that recombination between the flanking direct repeats occurs at a high frequency (10^-4) in vegetatively grown cultures. After excision, only one copy of the repeat sequence remains behind. Thus in the resulting strain, the Ura⁺ selection can be used again, either to disrupt a second gene in similar fashion or for another purpose.

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				Y. W. Jiang Transcriptional cosuppression of yeast Ty1 retrotransposons Genes & Dev., February 15, 2002; 16(4): 467 - 478. [Abstract] [Full Text] [PDF]

				P. A. Jauert, S. N. Edmiston, K. Conway, and D. T. Kirkpatrick RAD1 Controls the Meiotic Expansion of the Human HRAS1 Minisatellite in Saccharomyces cerevisiae Mol. Cell. Biol., February 1, 2002; 22(3): 953 - 964. [Abstract] [Full Text] [PDF]

				K. C. Abruzzi, A. Smith, W. Chen, and F. Solomon Protection from Free {beta}-Tubulin by the {beta}-Tubulin Binding Protein Rbl2p Mol. Cell. Biol., January 1, 2002; 22(1): 138 - 147. [Abstract] [Full Text] [PDF]

				G. Devasahayam, V. Chaturvedi, and S. D. Hanes The Ess1 Prolyl Isomerase Is Required for Growth and Morphogenetic Switching in Candida albicans Genetics, January 1, 2002; 160(1): 37 - 48. [Abstract] [Full Text] [PDF]

				J. F. Davidson and R. H. Schiestl Mitochondrial Respiratory Electron Carriers Are Involved in Oxidative Stress during Heat Stress in Saccharomyces cerevisiae Mol. Cell. Biol., December 15, 2001; 21(24): 8483 - 8489. [Abstract] [Full Text] [PDF]

				D. T. Scholes, M. Banerjee, B. Bowen, and M. J. Curcio Multiple Regulators of Ty1 Transposition in Saccharomyces cerevisiae Have Conserved Roles in Genome Maintenance Genetics, December 1, 2001; 159(4): 1449 - 1465. [Abstract] [Full Text] [PDF]

				N. Guttmann-Raviv, E. Boger-Nadjar, I. Edri, and Y. Kassir Cdc28 and Ime2 Possess Redundant Functions in Promoting Entry Into Premeiotic DNA Replication in Saccharomyces cerevisiae Genetics, December 1, 2001; 159(4): 1547 - 1558. [Abstract] [Full Text] [PDF]

				M. Bucholc, Y. Park, and A. J. Lustig Intrachromatid Excision of Telomeric DNA as a Mechanism for Telomere Size Control in Saccharomyces cerevisiae Mol. Cell. Biol., October 1, 2001; 21(19): 6559 - 6573. [Abstract] [Full Text] [PDF]

				M. Adam, F. Robert, M. Larochelle, and L. Gaudreau H2A.Z Is Required for Global Chromatin Integrity and for Recruitment of RNA Polymerase II under Specific Conditions Mol. Cell. Biol., September 15, 2001; 21(18): 6270 - 6279. [Abstract] [Full Text] [PDF]

				M. Bryk, M. Banerjee, D. Conte Jr., and M. J. Curcio The Sgs1 Helicase of Saccharomyces cerevisiae Inhibits Retrotransposition of Ty1 Multimeric Arrays Mol. Cell. Biol., August 15, 2001; 21(16): 5374 - 5388. [Abstract] [Full Text] [PDF]

				J. F. Davidson and R. H. Schiestl Cytotoxic and Genotoxic Consequences of Heat Stress Are Dependent on the Presence of Oxygen in Saccharomyces cerevisiae J. Bacteriol., August 1, 2001; 183(15): 4580 - 4587. [Abstract] [Full Text] [PDF]

				W. P. Voth, J. D. Richards, J. M. Shaw, and D. J. Stillman Yeast vectors for integration at the HO locus Nucleic Acids Res., June 15, 2001; 29(12): e59 - e59. [Abstract] [Full Text] [PDF]