System of Centromeric, Episomal, and Integrative Vectors Based on Drug Resistance Markers for
            <i>SacCharomyces Cerevisiae</i>

Taxis, Christof; Knop, Michael

doi:10.2144/000112040

Cited by 163 publications

(106 citation statements)

References 26 publications

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“…The presence of the mutation was confirmed by DNA sequencing. The ACT1 and act1-A167E inserts were subsequently excised from the pRS313 backbone by BamHI and SalI digestion and subcloned into pRS41H (hphNT1) (59).…”

Section: Frealign Refinementmentioning

confidence: 99%

Site-specific cation release drives actin filament severing by vertebrate cofilin

Kang

Bradley

Cao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this "stiffness cation" unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.cytoskeleton | persistence length | mechanics | electron cryomicroscopy A ctin polymerization powers the directed motility of eukaryotic cells and some pathogenic bacteria (1-3). Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. Sustained motility requires filament disassembly and subunit recycling. The essential regulatory protein cofilin severs actin filaments (4-6), which accelerates actin network reorganization by increasing the concentration of filament ends available for subunit exchange (7).Cofilin binding alters the structure and mechanical properties of filaments, which effectively introduces local "defects" that compromise filament integrity and promote severing (5). Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10-13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5,12,(14)(15)(16)(17)(18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21). These cation binding sites are referred to as "polymerization" and "stiffness" sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar f...

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Section: Frealign Refinementmentioning

confidence: 99%

Site-specific cation release drives actin filament severing by vertebrate cofilin

Kang

Bradley

Cao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Stable multi-copy integrations can be obtained by targeting the plasmids to abundant genomic sites, such as the long terminal repeats of Ty1 retrotransposons (δ sites) (Lee and Da Silva, 1997). So far, a vector series which integrates via double-crossover recombination is lacking (Voth et al, 2001;Taxis and Knop, 2006).…”

Section: Introductionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single-and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single-and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.

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“…dig1D represents the same genomic deletion as that of the dig1-D1THIS3 allele in JCY501; kss1D represents the same genomic deletion as that of the kss1DThisG allele in JCY110; ste12D represents the same genomic deletion as that of the ste12DTLEU2 allele in JCY512. The disruption markers KanMX4, HIS3MX6, CgHIS3, CgLEU2, CgTRP1, KlURA3, hphNT1, and natNT2 were amplified from pFA6a-KanMX4 (Wach et al 1994), pFA6a-HIS3MX6 (Wach et al 1997), pCgH, pCgL, pCgW, pKlU, pRS306H, and pRS306N (Taxis and Knop 2006), respectively. Unless otherwise indicated, strains were cultivated at 30°in standard rich (YP) or defined (SC) media (Burke et al 2000) containing 2% glucose (Glc)/ dextrose (D).…”

Section: Methodsmentioning

confidence: 99%

Systematic Epistasis Analysis of the Contributions of Protein Kinase A- and Mitogen-Activated Protein Kinase-Dependent Signaling to Nutrient Limitation-Evoked Responses in the Yeast Saccharomyces cerevisiae

Chen¹,

Thorner

2010

Genetics

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Cellular responses to environmental stimuli require conserved signal transduction pathways. In budding yeast (Saccharomyces cerevisiae), nutrient limitation induces morphological changes that depend on the protein kinase A (PKA) pathway and the Kss1 mitogen-activated protein kinase (MAPK) pathway. It was unclear to what extent and at what level there is synergy between these two distinct signaling modalities. We took a systematic genetic approach to clarify the relationship between these inputs. We performed comprehensive epistasis analysis of mutants lacking different combinations of all relevant pathway components. We found that these two pathways contribute additively to nutrient limitationinduced haploid invasive growth. Moreover, full derepression of either pathway rendered it individually sufficient for invasive growth and thus, normally, both are required only because neither is maximally active. Furthermore, in haploids, the MAPK pathway contributes more strongly than the PKA pathway to cell elongation and adhesion, whereas nutrient limitation-induced unipolar budding is independent of both pathways. In contrast, in diploids, upon nutrient limitation the MAPK pathway regulates cell elongation, the PKA pathway regulates unipolar budding, and both regulate cell adhesion. Thus, although there are similarities between haploids and diploids, cell type-specific differences clearly alter the balance of the signaling inputs required to elicit the various nutrient limitation-evoked cellular behaviors.

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System of Centromeric, Episomal, and Integrative Vectors Based on Drug Resistance Markers for SacCharomyces Cerevisiae

Cited by 163 publications

References 26 publications

Site-specific cation release drives actin filament severing by vertebrate cofilin

Site-specific cation release drives actin filament severing by vertebrate cofilin

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Systematic Epistasis Analysis of the Contributions of Protein Kinase A- and Mitogen-Activated Protein Kinase-Dependent Signaling to Nutrient Limitation-Evoked Responses in the Yeast Saccharomyces cerevisiae

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