Protein production in Saccharomyces cerevisiae for systems biology studies.

Proteins together with metabolites, nucleic acids, lipids, and other intracellular molecules form biological systems that involve networks of functional and physical interactions. To understand these interactions and the many other characteristics of proteins in the context of biochemical networks and systems biology, research aimed at studying medium and large sets of proteins is required. This either involves an investigation focused on individual protein activities in the mixture (e.g., cell extracts) or a protein characterization in the isolated form. This chapter provides an overview on the currently available resources and strategies for isolation of proteins from Saccharomyces cerevisiae. The use of standardized gene expression systems is discussed, and protein production protocols applied to the data generation pipeline for systems biology are described in detail.

Building a kinetic model of trehalose biosynthesis in Saccharomyces cerevisiae.

In this chapter, we describe the steps needed to create a kinetic model of a metabolic pathway based on kinetic data from experimental measurements and literature review. Our methodology is presented by utilizing the example of trehalose metabolism in yeast. The biology of the trehalose cycle is briefly reviewed and discussed.

Developmental regulation of an adhesin gene during cellular morphogenesis in the fungal pathogen Candida albicans.

Candida albicans expresses specific virulence traits that promote disease establishment and progression. These traits include morphological transitions between yeast and hyphal growth forms that are thought to contribute to dissemination and invasion and cell surface adhesins that promote attachment to the host. Here, we describe the regulation of the adhesin gene ALS3, which is expressed specifically during hyphal development in C. albicans. Using a combination of reporter constructs and regulatory mutants, we show that this regulation is mediated by multiple factors at the transcriptional level. The analysis of ALS3 promoter deletions revealed that this promoter contains two activation regions: one is essential for activation during hyphal development, while the second increases the amplitude of this activation. Further deletion analyses using the Renilla reniformis luciferase reporter delineate the essential activation region between positions -471 and -321 of the promoter. Further 5' or 3' deletions block activation. ALS3 transcription is repressed mainly by Nrg1 and Tup1, but Rfg1 contributes to this repression. Efg1, Tec1, and Bcr1 are essential for the transcriptional activation of ALS3, with Tec1 mediating its effects indirectly through Bcr1 rather than through the putative Tec1 sites in the ALS3 promoter. ALS3 transcription is not affected by Cph2, but Cph1 contributes to full ALS3 activation. The data suggest that multiple morphogenetic signaling pathways operate through the promoter of this adhesin gene to mediate its developmental regulation in this major fungal pathogen.

The relative merits of the tetO2 and tetO7 promoter systems for the functional analysis of heterologous genes in yeast and a compilation of essential yeast genes with tetO2 promoter substitutions.

We have generated a collection of yeast strains, each of which has an essential yeast gene under the control of the tetracycline-responsive, tetO, promoter. Screens using first-generation promoter-swap strains uncovered the non-specific responsiveness of the tetO7 promoter to a known human transcription factor (hIRF-1). Non-specific regulation was not observed with the tetO2 promoter. Reporter assays have been used to demonstrate this phenomenon. Subsequent efforts to generate a collection of tetracycline-regulatable strains have focused on the tetO2 promoter. These strains are available to the yeast community and can be used for functional genomics studies.

Doxycycline, the drug used to control the tet-regulatable promoter system, has no effect on global gene expression in Saccharomyces cerevisiae.

The tet-regulatable promoter system is commonly used for genetic studies in many eukaryotic organisms. The promoter is regulated using doxycycline. There are no obvious phenotypic effects observed when doxycycline is added to the growth medium of yeast to control expression from the promoter. It is widely accepted that doxycycline is innocuous to yeast. Global genetic studies are now commonplace and the tetO-system is being used in transcriptome studies. Hence, we wanted to ensure that the absence of phenotypic effects, on addition of doxycycline to the growth medium, is mirrored in transcriptome data. We have demonstrated that doxycycline has no significant effect on global transcription levels and will continue to use the tetO-regulatable promoter system for genetic studies.