New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast

Wosika, Victoria; Durandau, Eric; Varidel, Clémence; Aymoz, Delphine; Schmitt, Marta; Pelet, Serge

doi:10.1007/s00438-016-1249-1

Cited by 33 publications

(36 citation statements)

References 36 publications

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“…Each dPSTR is fully carried by a single integration vector (pSIV Wosika et al , ) and integrated in the genome. The red (and yellow) variants of the dPSTR (dPSTR R and dPSTR Y , respectively) are integrated in the URA3 (resp.…”

Section: Methodsmentioning

confidence: 99%

Timing of gene expression in a cell‐fate decision system

Aymoz

Solé

Pierre

et al. 2018

Molecular Systems Biology

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During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating‐responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contributes to determine the kinetics of transcription in a simplified cell‐fate decision system.

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Section: Methodsmentioning

confidence: 99%

Timing of gene expression in a cell‐fate decision system

Aymoz

Solé

Pierre

et al. 2018

Molecular Systems Biology

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“…The expression reporters pFIG1-qVenus and pSVS1-qVenus were cloned in pRS305 backbone and integrated in the LEU2 locus by digestion with ClaI3846. The MAPK activity reporter is cloned in a pSIVu vector and integrated in the URA3 locus47.…”

Section: Methodsmentioning

confidence: 99%

Nuclear relocation of Kss1 contributes to the specificity of the mating response

Pelet

2017

Sci Rep

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Mitogen Activated Protein Kinases (MAPK) play a central role in transducing extra-cellular signals into defined biological responses. These enzymes, conserved in all eukaryotes, exert their function via the phosphorylation of numerous substrates located throughout the cell and by inducing a complex transcriptional program. The partitioning of their activity between the cytoplasm and the nucleus is thus central to their function. Budding yeast serves as a powerful system to understand the regulation of these fundamental biological phenomena. Under vegetative growth, the MAPK Kss1 is enriched in the nucleus of the cells. Stimulation with mating pheromone results in a rapid relocation of the protein in the cytoplasm. Activity of either Fus3 or Kss1 in the mating pathway is sufficient to drive this change in location by disassembling the complex formed between Kss1, Ste12 and Dig1. Artificial enrichment of the MAPK Kss1 in the nucleus in presence of mating pheromone alters the transcriptional response of the cells and induces a cell-cycle arrest in absence of Fus3 and Far1.

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“…Unlike centromeric and multicopy 2µ plasmid, integrative plasmids guarantee a stable expression over time. However, multiple integration events leading to variations in plasmid copy number across selected clones are not uncommon (Wosika et al, 2016). This is due to the fact that the integration site is replicated following the first homologous recombination, hence enabling subsequent integrations ( Figure S1).…”

Section: Single Copy Integration Of Plasmids Using the Cosplay Toolboxmentioning

confidence: 99%

COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast

Goulev

Matifas

Heyer

et al. 2019

Preprint

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A large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.). Subsequently, these collections have been expanded by adding more diversity to many of the plasmid features, including new selection markers and new promoter sequences. However, the ever growing number of plasmid feature makes it unrealistic for research labs to maintain an up-to-date collection of plasmids.. Here, we developed the COSPLAY toolbox: a Golden Gate approach based on the scheme of a simple modular plasmid that recapitulates and completes all the properties of the pRS plasmids. The COSPLAY toolbox contains a basal collection of individual functional modules. Moreover we standardized a simple and rapid, software-assisted protocol which facilitates the addition of new personalized modules. Finally, our toolbox includes the possibility to select a genomic target location and to perform a single copy integration of the expression vector.

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New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast

Cited by 33 publications

References 36 publications

Timing of gene expression in a cell‐fate decision system

Timing of gene expression in a cell‐fate decision system

Nuclear relocation of Kss1 contributes to the specificity of the mating response

COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast

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