12.3: Experimental considerations
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You may be wondering why we are not using MET gene complementation to isolate transformants, since this is the goal of our semester’s project. There are several reasons why we are using YC-Ura plates, rather than YC-Met plates, to isolate tranformants. First, we need to ensure that the overexpression plasmids have successfully transformed the deletion strains and it is possible that the Met fusion proteins encoded by the plasmids will be unable to complement themet deficiencies in transformants. URA3 gene complementation offers a well-tested and reliable means to assess successful transformation that is independent of methionine metabolism.
A second issue relates to uncertainties associated with regulation of the plasmid ORFs
by the GAL1 promoter (Johnston, 1987). The GAL1 promoter is an inducible promoter that is normally repressed when cells are grown in glucose and induced when galactose replaces glucose as the carbon source. In its normal chromosomal location, the GAL1 promoter responds to a variety of positive and negative transcription regulators (Chapter 13). Although a large number of studies have established that the GAL1 promoter functions well in ectopic locations, such as plasmids, the promoter is not as tightly regulated in plasmids as in the yeast chromosome. Some of this difference may relate to copy number. Multiple copies of the pYES2.1 plasmids can be expected in tranformed cells.
Following the isolation of transformants of YC-Ura plates, you will analyze MET gene complementation on YC-Met plates containing either D-glucose and D-galacatose. Keep in mind that galactose and glucose may not function as simple “ON” and “OFF” switches because the regulatory balance is altered in transformed cells. It is possible, for example, that “leaky” gene transcription could occur in the presence of the normal repressor, D-glucose. In this case, METgenes would complement met mutants grown in D-glucose. It is also possible that transformed cells could produce excessive quantities of Met and proteins that are detrimental, or even fatal, to transformed cells.