A microarray-based protocol for monitoring the growth of yeast overexpression strains.
Gene overexpression can be used to investigate the biological pathways that are important in the response to a small molecule or other perturbation. To facilitate the use of gene overexpression in the study of small-molecule mechanisms, we developed a microarray-based protocol for monitoring the growth of a pool of yeast strains, each overexpressing a different protein. In this protocol, yeast harboring a set of approximately 3,900 galactose-inducible overexpression plasmids are grown in the absence or presence of a small molecule for multiple generations. The plasmids are then extracted from the two populations, processed and labeled in such a manner that their relative concentrations can be determined by competitive hybridization to a microarray. Although this protocol was developed for monitoring a specific set of overexpression plasmids, it could presumably be adapted to monitor yeast that have been transformed with any set of plasmids for which the gene inserts have been spotted, or otherwise arrayed, in a microarray format. This protocol can be completed in approximately 15 hours of hands-on time over the course of several days.
Butcher
Rebecca A
RA
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA. rebecca_butcher@hms.harvard.edu
Schreiber
Stuart L
SL
D005690:Galactose / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D040081:Protein Array Analysis / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism
D005690:Galactose