Citations Hub

Scheckhuber CQ, Wanger RA, Mignat CA, Osiewacz HD Cell Cycle. 2011 Sep 15;10(18):3105–10 Mitochondrial morphology is controlled by the opposing processes of fusion and fission. Previously, in baker's yeast it was shown that reduced mitochondrial fission leads to a network-like morphology, decreased sensitivity for the induction of apoptosis and a remarkable extension of both replicative and chronological lifespan. However, the effects of reduced mitochondrial fusion on aging are so far unknown and complicated by the fact that deletion of genes encoding components of mitochondrial fusion are often lethal to higher organisms. This is also true for the mammalian OPA1 protein, which is a key regulator of mitochondrial inner membrane fusion. Baker's yeast contains an OPA1 ortholog, Mgm1p. Deletion of Mgm1 is possible in yeast due to the fact that mitochondrial function is not essential for growth on glucose-containing media. In this study, we report that absence of mitochondrial fusion in the Δmgm1 mutant leads to a striking reduction of both replicative and chronological lifespan. Concomitantly, sensitivity to apoptosis elicitation via the reactive oxygen species hydrogen peroxide is substantially increased. These results demonstrate that the unopposed mitochondrial fission as displayed by the Δmgm1 mutant strongly affects organismal aging. Moreover, our results bear important clues for translational research to intervene into age-related degenerative processes also in multicellular organisms including humans.

Pérez-Través L, Lopes CA, Barrio E, Querol A Int J Food Microbiol. 2012 May 15;156(2):102–11 Several methods based on recombinant DNA techniques have been proposed for yeast strain improvement; however, the most relevant oenological traits depend on a multitude of loci, making these techniques difficult to apply. In this way, hybridization techniques involving two complete genomes became interesting. Natural hybrid strains between different Saccharomyces species have been detected in diverse fermented beverages including wine, cider and beer. These hybrids seem to be better adapted to fluctuating situations typically observed in fermentations due to the acquisition of particular physiological properties of both parental strains. In this work we evaluated the usefulness of three different hybridization methods: spore to spore mating, rare-mating and protoplast fusion for the generation of intra- and inter-specific stable hybrids, being the first report about the comparison of different methods to obtain artificial hybrids to be used in fermentations. Spore to spore mating is an easy but time-consuming method; hybrids generated with this technique could lack some of the industrially relevant traits present in the parental strains because of the segregation occurred during meiosis and spore generation prior to hybridization. Hybrids obtained by protoplast fusion get the complete information of both parents but they are currently considered as genetically modified organisms (GMOs). Finally, hybrids obtained by rare-mating are easily obtained by the optimized methodology described in this work, they originally contain a complete set of chromosomes of both parents and they are not considered as GMOs. Hybrids obtained by means of the three methodological approaches showed a high genetic variability; however, a loss ofgenetic material was detected in most of them. Based on these results, it became evident that a last crucial aspect to be considered in every hybridization program is the genetic stabilization of recently generated hybrids that guarantee its invariability during future industrial utilization. In this work, a wine yeast genetic stabilization process was developed and vegetatively stable hybrids were obtained.

Garcia B, Stollar EJ, Davidson AR Genetics. 2012 Aug;191(4):1199–211 Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conservedfeatures of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.

Tong AHY, Boone C Methods Mol Biol. 2006;313:171–92 Yeast/Methods Synthetic lethality occurs when the combination of two mutations leads to an inviable organism. Screens for synthetic lethal genetic interactions have been used extensively to identify genes whose products buffer one another or impinge on the same essential pathway. For the yeastSaccharomyces cerevisiae, we developed a method termed Synthetic Genetic Array (SGA) analysis, which offers an efficient approach for the systematic construction of double mutants and enables a global analysis of synthetic lethal genetic interactions. In a typical SGA screen, a query mutation is crossed to an ordered array of approx 5000 viable gene deletion mutants (representing approximately 80% of all yeast genes) such that meiotic progeny harboring both mutations can be scored for fitness defects. This array-based approach automates yeast genetic analysis in general and can be easily adapted for a number of different screens, including genetic suppression, plasmid shuffling, dosage lethality, or suppression.

Chang M, Parsons AB, Sheikh BH, Boone C, Brown GW Meth Enzymol. 2006;409:213–35 Yeast/Methods DNA damage response pathways have been studied extensively in the budding yeast Saccharomyces cerevisiae, yet new genes with roles in theDNA damage response are still being identified. In this chapter we describe the use of functional genomic approaches in the identification of DNAdamage response genes and pathways. These techniques take advantage of the S. cerevisiae gene deletion mutant collection, either as an ordered array or as a pool, and can be automated for high throughput.

Roguev A, Wiren M, Weissman JS, Krogan NJ Nat Methods. 2007 Oct;4(10):861-6. Epub 2007 Sep 23# Yeast/Methods Epistasis analysis, which reports on the extent to which the function of one gene depends on the presence of a second, is a powerful tool for studying the functional organization of the cell. Systematic genome-wide studies of epistasis, however, have been limited, with the majority of data being collected in the budding yeast, Saccharomyces cerevisiae. Here we present two 'pombe epistasis mapper' strategies, PEM-1 and PEM-2, which allow for high-throughput double mutant generation in the fission yeast, S. pombe. These approaches take advantage of a previously undescribed, recessive, cycloheximide-resistance mutation. Both systems can be used for genome-wide screens or for the generation of high-density, quantitative epistatic miniarray profiles (E-MAPs). Since S. cerevisiae and S. pombe are evolutionary distant, this methodology will provide insight into conserved biological pathways that are present in S. pombe, but not S. cerevisiae, and will enable a comprehensive analysis of the conservation of genetic interaction networks.

Tong AHY, Boone C Methods Mol Biol. 2007; 36:369-86,706-7 Yeast/Methods This chapter discusses an array-based synthetic lethal analysis approach, termed as “synthetic genetic array (SGA) analysis,” which is an automated method for constructing double mutants (or higher order allele combinations) and large-scale mapping of functional relationships between specific genes and pathways in yeast. In budding yeast, Saccharomyces cerevisiae, a complete set of gene deletion mutants has been constructed for each of the ˜6000 predicted genes in the genome, identifying ˜1000 essential genes and creating ˜5000 viable deletion mutants. The fact that over 80% of the predicted genes are not required for life reflects the robustness of biological circuits and may reflect cellular buffering against genetic variation. Hence, the collection of ˜5000 viable deletion mutants represents a valuable resource for systematic genetic analysis, providing the potential to examine 12.5 million different double-mutant combinations for a synthetic lethal or sick phenotype. When two genes show a synthetic lethal interaction, it often reflects that the gene products impinge on the same essential function, such that one pathway functionally compensates for, or buffers, the defects in the other. Thus, large-scale mapping of genetic interactions should provide a global view of functional relationships between genes and pathways.

Burston HE, Davey M, Conibear E Methods Mol Biol. 2008;457:29-39 Yeast/Methods The transport of membrane-bound proteins through post-Golgi compartments depends on the coordinated function of multiple genes that direct the recognition and routing of protein cargoes to their final cellular destination. As many of these sorting components are nonessential for viability,genome-wide screening of the yeast gene-deletion mutant collection provides a useful strategy for their identification. The potential of this approach is limited only by the availability of transport assays suitable for the high-throughput screening of yeast colony arrays. Two large-scale phenotypic screens to identify novel transport genes are described here. The fluorescence-based Calcofluor white assay identifies mutants with altered plasmamembrane localization of the chitin synthase Chs3, which recycles between the cell surface, endosomes, and the late Golgi. The carboxypeptidase Y (CPY) assay allows mutants of a distinct Golgi-to-vacuole transport pathway to be identified, due to the missorting and secretion of the vacuolar hydrolase CPY from the cell.

Wolinski H, Natter K, Kohlwein SD Methods Mol Biol. 2009;548:75–99 Yeast/Methods Despite its small size of 5-8 mum - only one order of magnitude above the wavelength of visible light - yeast has developed into an attractive system for light microscopic analysis. First, the ease of genetic manipulation and integrative transformation have opened numerous experimental strategies for genome-wide tagging approaches, e.g., with fluorescent proteins (as discussed in several chapters of this issue). Second, the large number of cells that can be simultaneously visualized provides an excellent basis for statistical image analysis, resulting in reliable morphological or localization information. Third, the flexibility of yeast cultivation in terms of biochemical manipulation, rapid cellular growth, mutant isolation or drug susceptibility offers an unprecedented spectrum of possibilities for in vivo functional studies, and analysis of cellular dynamics and organelle inheritance. Although yeast in itself is an interesting cellular system, its "prototype character" in understanding cellular metabolism, physiology, and signaling in eukaryotes accounts for its popular use in technology development and biomedical research.Here we discuss experimental strategies forlive yeast cell imaging, geared towards imaging-based large-scale screens. Major emphasis is on the methods for immobilizing cells under "physiological" conditions, with minimum impact on yeast. We also point out potential pitfalls resulting from live cell imaging that once again stresses the necessity for extremely careful experimental design and interpretation of data resulting from imaging experiments. It goes without saying that these problems are not restricted to yeast and are also highly relevant to "large" cells. If an image tells more than a thousand (perhaps misleading?) words, the ease of obtaining "images" thus rather suggests analyzing many thousands of images, to come up with one relevant and biologically significant conclusion.

Costanzo M, Boone C Methods Mol Biol. 2009;548:37–53 Yeast/Methods The development of genome-scale resources and high-throughput methodologies has enabled systematic assessment of gene function in vivo. Synthetic genetic array (SGA) analysis automates yeast genetic manipulation, permitting diverse analysis of approximately 5,000 viable deletion mutants in Saccharomyces cerevisiae. SGA methodology has enabled genome-wide synthetic lethal screening and construction of a large-scalegenetic interaction network for yeast. Genetic networks often reveal new components of specific pathways and functional relationships between genes whose products buffer one another or impinge on a common essential pathway. Because SGA analysis can be used to manipulate anygenetic element linked to a selectable marker, it is a highly versatile approach that can be adapted for a variety of different genetic screens, including synthetic lethality, dosage suppression, and dosage lethality. This chapter focuses on a specific SGA application for high-resolutiongenetic mapping, referred to as SGA mapping (SGAM), which enables the identification of suppressor mutations and thus provides a powerful means for interrogating gene function and pathway order.