Sassi HE, Bastajian N, Kainth P, Andrews BJ
Methods Mol Biol. 2009;548:55–73
Yeast/Methods
Temporal control of gene expression is a widespread feature of cell cycles, with clear transcriptional programs in bacteria, yeast, and metazoans. In budding yeast, approximately 1,000 genes are transcribed during a specific interval of the cell cycle. Although a number of factors that contribute to this periodic pattern of gene expression have been studied in Saccharomyces cerevisiae, pathways of cell cycle-regulated transcription remain largely undefined. To identify regulators of genes exhibiting cell cycle periodicity, we have developed a functional genomics approach termedreporter-based synthetic genetic array (R-SGA) analysis. Based on synthetic genetic array (SGA) analysis, R-SGA allows rapid and easily automated incorporation of a cell cycle reporter gene into the array of viable haploid yeast gene-deletion mutants. Scoring of reporter activity in mutant strains compared to wild type identifies candidate regulators of the cell cycle gene of interest. In contrast to microarrays, which generally provide information about the expression of all genes under a particular condition (for example, a single gene deletion), R-SGA analysis facilitates the study of the expression of a single gene in all deletion mutants. Our system can be adapted to examine the expression of any gene not only in the context of haploid deletion mutants but also using other array-based strain collections available to the yeast community.
Baryshnikova A, Costanzo M, Dixon S, Vizeacoumar FJ, Myers CL, Andrews B, Boone C
Meth Enzymol. 2010;470:145–79
Yeast/Methods
A genetic interaction occurs when the combination of two mutations leads to an unexpected phenotype. Screens for synthetic genetic interactions have been used extensively to identify genes whose products are functionally related. In particular, synthetic lethal genetic interactions often identify genes that buffer one another or impinge on the same essential pathway. For the yeast Saccharomyces cerevisiae, we developed a method termedsynthetic genetic array (SGA) analysis, which offers an efficient approach for the systematic construction of double mutants and enables a globalanalysis of synthetic genetic interactions. In a typical SGA screen, a query mutation is crossed to an ordered array of ~5000 viable gene deletion mutants (representing ~80% of all yeast genes) such that meiotic progeny harboring both mutations can be scored for fitness defects. This approach can be extended to all ~6000 genes through the use of yeast arrays containing mutants carrying conditional or hypomorphic alleles of essential genes. Estimating the fitness for the two single mutants and their corresponding double mutant enables a quantitative measurement of geneticinteractions, distinguishing negative (synthetic lethal) and positive (within pathway and suppression) interactions. The profile of genetic interactions represents a rich phenotypic signature for each gene and clustering genetic interaction profiles group genes into functionally relevant pathways and complexes. This array-based approach automates yeast genetic analysis in general and can be easily adapted for a number of different geneticscreens or combined with high-content screening systems to quantify the activity of specific reporters in genome-wide sets of single or more complex multiple mutant backgrounds. Comparison of genetic and chemical-genetic interaction profiles offers the potential to link bioactive compounds to their targets. Finally, we also developed an SGA system for the fission yeast Schizosaccharomyces pombe, providing another model system for comparative analysis of genetic networks and testing the conservation of genetic networks over millions of years of evolution.
Cohen Y, Schuldiner M
Methods Mol Biol. 2011;781:127–59
Yeast/Methods
High-throughput methodologies have created new opportunities for studying biological phenomena in an unbiased manner. Using automated cell manipulations and microscopy platforms, it is now possible to easily screen entire genomes for genes that affect any cellular process that can be visualized. The onset of these methodologies promises that the near future will bring with it a more comprehensive and richly integrated understanding of complex and dynamic cellular structures and processes. In this review, we describe how to couple systematic genetic tools in the budding yeast Saccharomyces cerevisiae alongside robotic visualization systems to attack biological questions. The combination of high-throughputmicroscopy screens with the powerful, yet simple, yeast model system for studying the eukaryotic cell should pioneer new knowledge in all areas of cell biology.
Khmelinskii A, Meurer M, Duishoev N, Delhomme N, Knop M
PLoS One. 2011;6(8):e23794
Yeast/Methods
Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.
Dünkler A, Müller J, Johnsson N
Methods Mol Biol. 2012;786:115–30
Yeast/Methods
A detailed understanding of a cellular process requires the knowledge about the interactions between its protein constituents. The Split-Ubiquitintechnique allows to monitor and detect interactions of very diverse proteins, including transcription factors and membrane-associated proteins. The technique is based on unique features of ubiquitin, the enzymes of the ubiquitin pathway, and the reconstitution of a native-like ubiquitin from its N- and C-terminal fragments. Using Ura3p as a reporter for the reconstitution of the ubiquitin fragments, methods are presented that enable to screen in yeast for interaction partners of a given protein with either a randomly generated expression library or a defined but more limited array of protein fusions.
Bean GJ, Jaeger PA, Bahr S, Ideker T
PLoS One. 2014 Jan 21;9(1):e85177
Yeast/Methods
High-throughput genetic screens in model microbial organisms are a primary means of interrogating biological systems. In numerous cases, such screens have identified the genes that underlie a particular phenotype or a set of gene-gene, gene-environment or protein-protein interactions, which are then used to construct highly informative network maps for biological research. However, the potential test space of genes, proteins, or interactions is typically much larger than current screening systems can address. To push the limits of screening technology, we developed an ultra-high-density, 6144-colony arraying system and analysis toolbox. Using budding yeast as a benchmark, we find that these tools boost geneticscreening throughput 4-fold and yield significant cost and time reductions at quality levels equal to or better than current methods. Thus, the newultra-high-density screening tools enable researchers to significantly increase the size and scope of their genetic screens.
Khmelinskii A, Knop M
Methods Mol Biol. 2014;1174:195-210
Yeast/Methods
Fluorescent timers (FTs) are fluorescent proteins that change color with time. FTs can be used as tags to follow protein dynamics in living cells. Recently we described a novel class of FTs called tandem fluorescent protein timers (tFTs). Each tFT is a tandem fusion of two different conventional fluorescent proteins having distinct kinetics of fluorophore maturation. tFTs suitable for studying protein dynamics on different scales can be generated from a broad range of commonly used fluorescent proteins. Here we describe how to establish new tFTs and consider potential pitfalls. We detail a protocol for quantitative fluorescence microscopy imaging and analysis of intracellular protein dynamics with tFTs in the budding yeast Saccharomyces cerevisiae.
Braberg H, Alexander R, Shales M, Xu J, Franks-Skiba KE, Wu Q, Haber JE, Krogan NJ
Nat Protoc. 2014 Aug;9(8):1867–81
Yeast/Methods
The quantitative analysis of genetic interactions between pairs of gene mutations has proven to be effective for characterizing cellular functions, but it can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed triple-mutantanalysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, which is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principal actors are deleted. TMA has also uncovered double-mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete and measuresinteractions for up to 30 double mutants against a library of 1,536 single mutants.
Dalton L, Davey M, Conibear E
Methods Mol Biol. 2015;1270:395-409
Yeast/Methods
Transport of membrane proteins between cellular organelles requires the concerted action of many regulatory factors, which aid in cargo recognition and vesicle formation, targeting, and fusion. The yeast Saccharomyces cerevisiae is a useful model system for studying such regulators, due to the availability of genome-wide mutant collections and reporter proteins that provide sensitive biochemical readouts of individual transport pathways. Here, we describe an enzymatic invertase assay for evaluating endocytic recycling using a chimeric GFP-Snc1-Suc2 reporter. Cell surface levels of this reporter can be measured by a colorimetric assay that monitors sucrose hydrolysis at the plasma membrane, using two different methods. The first is a semiquantitative agar overlay assay followed by image densitometry that is suitable for high-throughput screening of arrayed yeast colonies. In the second, more quantitative assay, an enzymatic solution is added to yeast cultures in a multi-well plate and the absorbance is assessed by a plate reader. Furthermore, the modular nature of the chimeric reporter allows alternate transport signals to be introduced, thereby expanding the range of transport pathways that can be evaluated by this method. Together these techniques can be used to explore the function of genes involved in a variety of cellular trafficking pathways.