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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