Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae

Longtine, Mark S.; Mckenzie, Amos; DeMarini, Douglas J.; Shah, Nirav G.; Wach, Achim; Brachat, Arndt; Philippsen, Peter; Pringle, John R.

doi:10.1002/(sici)1097-0061(199807)14:10<953::aid-yea293>3.3.co;2-l

Cited by 509 publications

(672 citation statements)

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“…Yeast Nap1 proteins were expressed and purified similar to described previously (McBryant et al, 2003), except that they contained three (C200A, C249A, C272A) or four (plus C414A) mutations and were from a pBAT4 or pHAT4 plasmid respectively. NAP1 was deleted in the parental yeast strain bar1Δ ( MAT α, his3 Δ 1, ura3 Δ 0, leu2 Δ 0, met15 Δ 0, bar1Δ::KanMX4 ) (Open Biosystems) using established protocols (Longtine et al, 1998). Unlabeled and 5′ Atto 647N-labeled 30mer oligos were purchased from EGT.…”

Section: Methodsmentioning

confidence: 99%

Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

et al. 2013

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Summary The histone H2A-H2B heterodimer is an integral component of the nucleosome. The cellular localization and deposition of H2A-H2B into chromatin is regulated by numerous factors including histone chaperones such as Nucleosome Assembly Protein 1 (Nap1). We use hydrogen-deuterium exchange coupled to mass spectrometry to characterize H2A-H2B and Nap1. Unexpectedly, we find that at low ionic strength the α-helices in H2A-H2B are frequently sampling partially disordered conformations, and that binding to Nap1 reduces this conformational sampling. We identify the interaction surface between H2A-H2B and Nap1, and confirm its relevance both in vitro and in vivo. We show that two copies of H2A-H2B bound to a Nap1 homodimer form a tetramer with contacts between H2B chains similar to those in the four-helix bundle structural motif. The organization of the complex reveals that Nap1 competes with histone-DNA and inter-histone interactions observed in the nucleosome, thereby regulating the availability of histones for chromatin assembly.

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Section: Methodsmentioning

confidence: 99%

Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

et al. 2013

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“…Plasmids pFA6a‐GFP‐KanMX6 (Longtine et al., 1998) and pBS34‐mCherry‐KanMX6 (Hailey, Davis, & Muller, 2002) were digested with NotI and used as template for PCR (Yeast Resource Center, University of Washington). For some strains, the antibiotic resistance in fluorescently labeled yeast was subsequently switched via transformation with NatMX or HphMX (Table S1).…”

Section: Methodsmentioning

confidence: 99%

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Deschaine

Heysel

Lenhart

et al. 2018

Ecology and Evolution

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Microbes can engage in social interactions ranging from cooperation to warfare. Biofilms are structured, cooperative microbial communities. Like all cooperative communities, they are susceptible to invasion by selfish individuals who benefit without contributing. However, biofilms are pervasive and ancient, representing the first fossilized life. One hypothesis for the stability of biofilms is spatial structure: Segregated patches of related cooperative cells are able to outcompete unrelated cells. These dynamics have been explored computationally and in bacteria; however, their relevance to eukaryotic microbes remains an open question. The complexity of eukaryotic cell signaling and communication suggests the possibility of different social dynamics. Using the tractable model yeast, Saccharomyces cerevisiae, which can form biofilms, we investigate the interactions of environmental isolates with different social phenotypes. We find that biofilm strains spatially exclude nonbiofilm strains and that biofilm spatial structure confers a consistent and robust fitness advantage in direct competition. Furthermore, biofilms may protect against killer toxin, a warfare phenotype. During biofilm formation, cells are susceptible to toxin from nearby competitors; however, increased spatial use may provide an escape from toxin producers. Our results suggest that yeast biofilms represent a competitive strategy and that principles elucidated for the evolution and stability of bacterial biofilms may apply to more complex eukaryotes.

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“…One copy of the DUS2, MOD5, RPA49 , and TRM140 genes was independently deleted in the diploid strain UMY2366 by a PCR-mediated strategy [59,60]. The generated heterozygous diploids were allowed to sporulate and the dus2Δ (UMY2872), mod5Δ (UMY2565), rpa49Δ (MJY1111) and trm140Δ (MJY1110) strains obtained from tetrads.…”

Section: Methodsmentioning

confidence: 99%

Identification of factors that promote biogenesis of tRNA_CGA^Ser

Zhou

Byström

et al. 2018

RNA Biology

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A wide variety of factors are required for the conversion of pre-tRNA molecules into the mature tRNAs that function in translation. To identify factors influencing tRNA biogenesis, we previously performed a screen for strains carrying mutations that induce lethality when combined with a sup61-T47:2C allele, encoding a mutant form of . Analyzes of two complementation groups led to the identification of Tan1 as a protein involved in formation of the modified nucleoside N4-acetylcytidine (ac4C) in tRNA and Bud13 as a factor controlling the levels of ac4C by promoting TAN1 pre-mRNA splicing. Here, we describe the remaining complementation groups and show that they include strains with mutations in genes for known tRNA biogenesis factors that modify (DUS2, MOD5 and TRM1), transport (LOS1), or aminoacylate (SES1) . Other strains carried mutations in genes for factors involved in rRNA/mRNA synthesis (RPA49, RRN3 and MOT1) or magnesium uptake (ALR1). We show that mutations in not only DUS2, LOS1 and SES1 but also in RPA49, RRN3 and MOT1 cause a reduction in the levels of the altered . These results indicate that Rpa49, Rrn3 and Mot1 directly or indirectly influence biogenesis.

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Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae

Cited by 509 publications

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Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Identification of factors that promote biogenesis of tRNA_CGA^Ser

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Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae

Cited by 509 publications

References 0 publications

Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

Chaperone Nap1 Shields Histone Surfaces Used in a Nucleosome and Can Put H2A-H2B in an Unconventional Tetrameric Form

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

Identification of factors that promote biogenesis of tRNACGASer

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Identification of factors that promote biogenesis of tRNA_CGA^Ser