Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Z, Li; Vizeacoumar, Frederick S.; Bahr, Sondra; Li, Jingjing; Warringer, Jonas; Min, Renqiang; VanderSluis, Benjamin; Bellay, Jeremy; DeVit, Michael J.; Fleming, James; Stephens, Andrew D.; Haase, Julian; Lin, Zhen Yuan; Baryshnikova, Anastasia; Lü, Hong; Yan, Zhun; Jin, Ke; Barker, Sarah L.; Datti, Alessandro; Giaever, Guri; Nislow, Corey; Bulawa, Chris; Myers, Chad L.; Costanzo, Michael; Gingras, Anne‐Claude; Zhang, Zhaolei; Bloom, Kerry; Andrews, Brenda; Boone, Charles

doi:10.1038/nbt.1832

Cited by 379 publications

(379 citation statements)

References 56 publications

Supporting

Mentioning

360

Contrasting

Unclassified

Order By: Relevance

“…48 cdc3(G365R) mutants were CBY07236 (BY4741 cdc3 (G365R)::kanMX) 49 and M-1622 (YEF473 MATa cdc3(G365R)). 50 The whi3D::kanMX and swe1D::kanMX derivatives of BY4741 or its MATa MET17C lys2D relative BY4742 were obtained from the haploid deletion collections 51 and verified by PCR-amplifying and sequencing the "barcodes" flanking the gene replacement alleles.…”

Section: Methodsmentioning

confidence: 99%

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

et al. 2016

View full text Add to dashboard Cite

Septin proteins form highly conserved cytoskeletal filaments composed of hetero-oligomers with strict subunit stoichiometry. Mutations within one hetero-oligomerization interface (the "G" interface) bias the mutant septin toward conformations that are incompatible with filament assembly, causing disease in humans and, in budding yeast cells, temperature-sensitive defects in cytokinesis. We previously found that, when the amount of other hetero-oligomerization partners is limiting, wild-type and G interfacemutant alleles of a given yeast septin "compete" along parallel but distinct folding pathways for occupancy of a limited number of positions within septin hetero-octamers. Here, we synthesize a mathematical model that outlines the requirements for this phenomenon: if a wild-type septin traverses a folding pathway that includes a single rate-limiting folding step, the acquisition by a mutant septin of additional slow folding steps creates an initially large disparity between wild-type and mutant in the cellular concentrations of oligomerization-competent monomers. When the 2 alleles are co-expressed, this kinetic disparity results in mutant exclusion from hetero-oligomers, even when the folded mutant monomer is oligomerization-competent. To test this model experimentally, we first visualize the kinetic delay in mutant oligomerization in living cells, and then narrow or widen the "window of opportunity" for mutant septin oligomerization by altering the length of the G1 phase of the yeast cell cycle, and observe the predicted exacerbation or suppression, respectively, of mutant cellular phenotypes. These findings reveal a fundamental kinetic principle governing in vivo assembly of multiprotein complexes, independent of the ability of the subunits to associate with each other.

show abstract

Section: Methodsmentioning

confidence: 99%

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

et al. 2016

View full text Add to dashboard Cite

show abstract

“…The XRN1 gene was deleted from the rlg1-4 strain by gene replacement with a bacterial hygromycin B resistance gene (Hph) to construct rlg1-4 xrn1D. The ts rat1-1 strain was a gift from Dr. Charlie Boone (Li et al 2011).…”

Section: Yeast Strainsmentioning

confidence: 99%

“…To address these issues, we examined whether Rat1, the nuclear 59-to-39 exonuclease Xrn1 ortholog (Johnson 1997), affects intron turnover. We used a ts mutant of RAT1, rat1-1 (Li et al 2011), since RAT1 is an essential gene that is not contained in the deletion collection. Similar to wild type (Fig.…”

Section: The Trna Ligase Rlg1 Phosphorylates Trna Introns Prior To Dementioning

confidence: 99%

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

Hopper

2014

Genes Dev.

View full text Add to dashboard Cite

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although~600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 59 termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 59-to-39 exonuclease Xrn1, which has specificity for RNAs with 59 monophosphate.

show abstract

“…Phenomics requires the availability of large cohorts of populations of genetically diverse strains, with the model yeast Saccharomyces cerevisiae leading the way. Collections of artificial gene constructs, e.g., single gene knockout or overexpression strains, have facilitated gene function analysis in lab strains (Giaever et al 2002;Sopko et al 2006;Li et al 2011). Large crosses of natural, industrial, or clinical isolates allow dissection of the genotype-phenotype relationship in wider perspectives (Cubillos et al , 2013Parts et al 2011;Bloom et al 2013).…”

mentioning

confidence: 99%

Scan-o-matic: high-resolution microbial phenomics at a massive scale

Zackrisson

Hallin²,

Ottosson

et al. 2015

Preprint

Self Cite

View full text Add to dashboard Cite

The capacity to map traits over large cohorts of individuals-phenomics-lags far behind the explosive development in genomics. For microbes, the estimation of growth is the key phenotype because of its link to fitness. We introduce an automated microbial phenomics framework that delivers accurate, precise, and highly resolved growth phenotypes at an unprecedented scale. Advancements were achieved through the introduction of transmissive scanning hardware and software technology, frequent acquisition of exact colony population size measurements, extraction of population growth rates from growth curves, and removal of spatial bias by reference-surface normalization. Our prototype arrangement automatically records and analyzes close to 100,000 growth curves in parallel. We demonstrate the power of the approach by extending and nuancing the known salt-defense biology in baker's yeast. The introduced framework represents a major advance in microbial phenomics by providing high-quality data for extensive cohorts of individuals and generating well-populated and standardized phenomics databases KEYWORDS

show abstract

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Cited by 379 publications

References 56 publications

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

Scan-o-matic: high-resolution microbial phenomics at a massive scale

Contact Info

Product

Resources

About