Abstract
The GAL genetic switch of Saccharomyces cerevisiae exhibits an ultrasensitive response to the inducer galactose as well as the "all-or-none" behavior characteristic of many eukaryotic regulatory networks. We have constructed a strain that allows intermediate levels of gene expression from a tunable GAL1 promoter at both the population and the single cell level by altering the regulation of the galactose permease Gal2p. Similar modifications to other feedback loops regulating the Gal80p repressor and the Gal3p signaling protein did not result in similarly tuned responses, indicating that the level of inducer transport is unique in its ability to control the switch response of the network. In addition, removal of the Gal1p galactokinase from the network resulted in a regimed response due to the dual role of this enzyme in galactose catabolism and transport. These two activities have competing effects on the response of the network to galactose such that the transport effects of Gal1p are dominant at low galactose concentrations, whereas its catabolic effects are dominant at high galactose concentrations. In addition, flow cytometry analysis revealed the unexpected phenomenon of multiple populations in the gal1delta strains, which were not present in the isogenic GAL1 background. This result indicates that Gal1p may play a previously undescribed role in the stability of the GAL network response.
Highlights
The response of the GAL network was determined when the outermost positive feedback loop controlling the autocatalytic expression of the galactose permease Gal2p was removed
The data from the population-averaged transcriptional activation assays demonstrate that the positive feedback loop regulating the expression of Gal2p is necessary for the sharp, autocatalytic response of the system to galactose observed in the wild-type strain
We propose that under these conditions, transport effects limit the intracellular galactose concentration and the ensuing network response
Summary
Yeast Strain Construction—The wild-type haploid yeast strain used in this study is W303␣ (MATa his trp leu ura ade). A cassette harboring an E. coli kanamycin resistance gene and associated promoter and terminator elements with ends homologous to regions flanking GAL2 on the chromosome was constructed by amplifying the appropriate segment from pFA6a-ZZ-TEV-S-kanMX6 [20]. A second cassette harboring the tetO2 response element and minimal CYC1 promoter, the tTA transactivator and associated promoter and terminator elements, and the kanamycin resistance gene and associated promoter and terminator elements with ends homologous to regions flanking the GAL2 promoter was constructed in two steps (Fig. 1C). A third cassette harboring a Schizosaccharomyces pombe histidine biosynthetic gene (his5ϩ) and associated promoter and terminator elements with ends homologous to regions flanking GAL1 on the chromosome was constructed by amplifying the appropriate segment from pFA6-S-TEV-ZZ-HIS3MX6 [20]. All other cassettes were constructed with standard PCR procedures in a Dyad PCR machine (MJ Research) with TaqDNA
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