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. Author manuscript; available in PMC: 2010 Nov 15.
Published in final edited form as: J Am Chem Soc. 2008 Dec 24;130(51):17446–17452. doi: 10.1021/ja8055744

Figure 2.

Figure 2

Chemical complementation detects bond cleavage reactions A. In the cellulase growth selection, the Methotrexate-Cellotetraose-Dexamethasone (Mtx-Cel-Dex) heterodimer links a DNA-binding domain fused to dihydrofolate reductase (DBD-DHFR) and an activation domain fused to the glucocorticoid receptor (AD-GR), activating transcription of the toxic URA3 reporter gene, thus leading to cell death. Active cellulases cleave the β-1,4-glucosidic bond in the Mtx-Cel-Dex linker, decreasing transcription of the toxic URA3 reporter gene, and leading to cell survival. B. Structure of the Mtx-Cel-Dex chemical inducer of dimerization used for this assay. C. The URA3 counter selection strain was constructed in three steps. First, the URA3 gene was introduced at the ura3-52 locus of the chemical complementation yeast strain (FY251) via homologous recombination. Next, the AD-GR and the DBD-DHFR constructs were introduced into FY251 via transformation. Finally, the LexA DNA binding site and the tightly regulated Spo13 promoter were introduced upstream of the URA3 reporter gene via homologous recombination.