Abstract
Clonal selection strategies are central tools in molecular biology. We developed a general strategy to dissect protein functions through positive and negative clonal selection for protein-protein interactions, based on a protein-fragment complementation assay using Saccharomyces cerevisiae cytosine deaminase as a reporter. We applied this method to mutational or chemical disruption of protein-protein interactions in yeast and to dissection of the functions of an allosterically activated transcription factor, Swi6.
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Acknowledgements
We thank H. Bussey (McGill University) for providing yeast strains and p41XGal1 plasmids, B. Andrews (Donelly Centre, University of Toronto) for plasmids pBA487 and pBA251 and Swi6 antibodies, T. Clackson and V. Rivera (Ariad) for pC4EN-FM3, M.J. Booth for constructing p415Gal-Linker-OyCD-F[2], and M. Malleshaiah and C. Chan for comments. This research was supported by grants from the Canadian Institutes of Health Research (MOP-152556) and the Canada Research Chairs Program to S.W.M. P.H.E. acknowledges the Faculté des etudes supérieurs de l'Université de Montréal for scholarships.
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P.H.E. and S.W.M. designed the experiments, analyzed the results and wrote the manuscript. P.H.E. performed the experiments.
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Ear, P., Michnick, S. A general life-death selection strategy for dissecting protein functions. Nat Methods 6, 813–816 (2009). https://doi.org/10.1038/nmeth.1389
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DOI: https://doi.org/10.1038/nmeth.1389
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