Identification of Protein–DNA Interactions Using Enhanced Yeast One-Hybrid Assays and a Semiautomated Approach
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Two-hybrid screening
Identification
Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non‐artificial conditions
Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA. These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell. Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB. The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events. Copyright © 2000 John Wiley & Sons, Ltd.
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The GAL promoters are applied in metabolic engineering and synthetic biology to control gene expression in the budding yeast Saccharomyces cerevisiae. In gal80Δ background strains, they show diauxie-inducible expression, a feature beneficial in metabolic pathway optimization. However, only a limited number of GAL promoters have been characterized and are available for engineering purposes. Multiple uses of the same promoters can result in genetic instability in engineered strains due to homologous recombination. Here, 11 GAL1/2 promoters from other Saccharomyces species were isolated and characterized in S. cerevisiae. They exhibited diauxie-inducible expression patterns with low strength in exponential growth phase and induction in the ethanol growth phase. These promoters represent an expansion to the collection of GAL promoters available for genetic engineering in S. cerevisiae, including an increased diversity of expression levels. This provides the capacity for increased numbers of genetic manipulations with a lower risk of genetic instability.
Heterologous
Heterologous expression
Metabolic Engineering
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This protocol outlines a method for extracting total lipids from Baker's yeast, Saccharomyces cerevisiae. It has been adapted from Roy et al., J. Lipid Res. 2018. doi: 10.1194/jlr.M088559.
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Two-hybrid screening
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Two-hybrid screening
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The yeast two-hybrid system is a sensitive method to detect intracellular interactions between proteins. It has been successfully employed in the identification of novel interactors of several proteins, including adhesion molecules (for a general review on the method, see refs. 1 and 2). In general, the high sensitivity of the yeast two-hybrid system allows the detection of weak and transient interactions that escape biochemical analysis. Howewer, in some limited cases, interactions found with biochemical methods have not been reproduced in the yeast assay, possibly because of the lack of some posttranslational modifications. More generally, the high sensitivity of the yeast interaction assay may lead to the disclosure of false-positive protein-protein interactions. For this reason, it is always necessary to confirm the yeast interaction data using other methods of study for protein binding.
Two-hybrid screening
Identification
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One of the key feature of prions is the ability to be stable in two alternative conformations. Besides the intensively studied mammalian prions, there are also prion proteins present in the yeast Saccharomyces cerevisiae. Research in this field has lead to opposite hypotheses that explain the sense of presence of [PSI+] prion in yeast cells. Some authors postulate e of role of the prions in the evolution of S. cerevisiae, whereas other investigators point out the negative influence of these particles upon the yeast physiology. In recent years, yeast prions are used for anti-prion drug screening, because of common features with mammalian prions. This work presents the most intensively studied fields of the research carried out on [PSI+] prion in yeast.
Prion Proteins
Fungal prion
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Ethyl methanesulfonate
Methyl methanesulfonate
Strain (injury)
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