Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells

Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations

Yeast Two-Hybrid: State of the Art

Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination

Quantitative β-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system

Measurement of β-galactosidase (β-gal) activity is an important step in every yeast two-hybrid assay, yet many commonly used methods have distinct disadvantages, such as being only qualitative, time-consuming, and cumbersome when processing large numbers of samples. To overcome these drawbacks, we have implemented a novel technique, termed pellet X-gal assay, that allows simultaneous quantitative measurements from large numbers of samples with a minimum of hands-on time. The method was tested using five different, previously described protein-protein interactions and compared to two standard methods, the colony filter lift and the liquid ONPG assay. Our assay allows accurate quantitative measurements of protein-protein interactions and covers a greater dynamic range than the classic ONPG assay. The novel assay is robust and requires very little handling, making it suitable for applications in which several hundreds of individual protein interaction pairs need to be measured simultaneously