A fitness-based interferential genetics approach using hypertoxic/inactive gene alleles as references
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Genetics, genomics, and biochemistry have all been of immense help in characterizing macromolecular cell entities and their interactions. Still, obtaining an overall picture of the functioning of even a simple unicellular species has remained a challenging task. One possible way to obtain a comprehensive picture has been described: by capitalizing on the observation that the overexpression on a multicopy plasmid of apparently any wild-type gene in yeast can lead to some negative effect on cell fitness (referring to the concept of “gene toxicity”), the FIG (fitness-based interferential genetics) approach was devised for selecting normal genes that are in antagonistic (and potentially also agonistic) relationship with a particular gene used as a reference. Herein, we take a complementary approach to FIG, by first selecting a “hypertoxic” allele of the reference gene—which easily provides the general possibility of obtaining gene products with the remarkable property of being inactive without altering their macromolecular interactivity—and then looking for the genes that interact functionally with this reference. Thus, FIG and the present approach (Trap-FIG), both taking advantage of the negative effects on cell fitness induced by various quantitative modulations in cellular networks, could potentially pave the way for the emergence of efficient in situ biochemistry.
KeywordsFitness-based interferential genetics Ino4 transcription factor Macromolecular network Protein kinase C Interactive-inactive allele
We thank D. Levin for strain DL106 and G. Paravicini (Glaxo) for plasmid pGEM7-PKC1. This work was supported by the Centre National de la Recherche Scientifique (France).
- Gardner R, Putnam CW, Weinert T (1999) RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast. EMBO J 18:3178–3185Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Sherman F, Fink G, Hicks J (1987) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar