yeast genetics
Recently Published Documents


TOTAL DOCUMENTS

179
(FIVE YEARS 15)

H-INDEX

16
(FIVE YEARS 1)

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Dorota Rousova ◽  
Vaishnavi Nivsarkar ◽  
Veronika Altmannova ◽  
Vivek B Raina ◽  
Saskia K Funk ◽  
...  

In meiosis, DNA double strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific “axis-tethered loop” chromosome organization. Through in vitro reconstitution and budding yeast genetics we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates to nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.


2021 ◽  
Author(s):  
Andrew D Bailey ◽  
Jason Talkish ◽  
Hongxu Ding ◽  
Haller Igel ◽  
Alejandra Duran ◽  
...  

Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites, within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.


2021 ◽  
Author(s):  
Robin Ganesan ◽  
Kotchaphorn Mangkalaphiban ◽  
Richard E. Baker ◽  
Feng He ◽  
Allan Jacobson

SUMMARYUpf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.


2021 ◽  
Author(s):  
Satoshi Okada ◽  
Goro Doi ◽  
Shitomi Nakagawa ◽  
Emiko Kusumoto ◽  
Takashi Ito

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.


Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 945
Author(s):  
James E. Haber

Miroslav Radman’s far-sighted ideas have penetrated many aspects of our study of the repair of broken eukaryotic chromosomes. For over 35 years my lab has studied different aspects of the repair of chromosomal breaks in the budding yeast, Saccharomyces cerevisiae. From the start, we have made what we thought were novel observations that turned out to have been predicted by Miro’s extraordinary work in the bacterium Escherichia coli and then later in the radiation-resistant Dienococcus radiodurans. In some cases, we have been able to extend some of his ideas a bit further.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Valeria Zoni ◽  
Rasha Khaddaj ◽  
Pablo Campomanes ◽  
Abdou Rachid Thiam ◽  
Roger Schneiter ◽  
...  

Cells store energy in the form of neutral lipids (NLs) packaged into micrometer-sized organelles named lipid droplets (LDs). These structures emerge from the endoplasmic reticulum (ER) at sites marked by the protein seipin, but the mechanisms regulating their biogenesis remain poorly understood. Using a combination of molecular simulations, yeast genetics, and fluorescence microscopy, we show that interactions between lipids’ acyl-chains modulate the propensity of NLs to be stored in LDs, in turn preventing or promoting their accumulation in the ER membrane. Our data suggest that diacylglycerol, which is enriched at sites of LD formation, promotes the packaging of NLs into LDs, together with ER-abundant lipids, such as phosphatidylethanolamine. On the opposite end, short and saturated acyl-chains antagonize fat storage in LDs and promote accumulation of NLs in the ER. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.


2021 ◽  
Author(s):  
Harry Singer ◽  
Terrance G Cooper

Abstract Micromanipulators, more than any other instrument, opened the early doors to developing the powerful genetics of yeast that underlies much of the molecular work today. The ability to separate the spores of a tetrad and analyze their phenotypes generated the genetic maps and biology upon which subsequent cloning, sequencing, cutting edge molecular and cell biology depended. This work describes the development of those micromanipulators from garage to barn to factory and the developer of the sophisticated instruments we use today. For more than 30 years Carl Singer and his family were staunch and generous supporters of the International Conferences on Yeast Genetics and Molecular Biology meetings both in Europe and America. Carl Singer's displays at meetings became a traditional fixture and engaged the appetites of many students and advanced researchers to employ a technique that many perceived as too complicated or difficult, but which he made simple and easy to learn. His experiences also document a sketch of the international yeast meetings, their venues and how they developed through the years.


2020 ◽  
Author(s):  
Haina Huang ◽  
Katrin Karbstein

AbstractRibosomes are among the largest folded RNAs, whose function depends on their structure. Nonetheless, in vitro studies indicate a propensity of rRNAs to misfold. We use a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics to dissect the final RNA folding steps of the small ribosomal subunit head. Our data demonstrate how a succession of assembly factors prevent the early folding of an RNA loop, thereby allowing an adjacent helical junction to fold first. Deletion of the assembly factors to allow the loop to fold first traps a misfolded junction, demonstrating how assembly factors modulate the folding pathway to enable challenging motifs to fold independent of their interaction partners and guide folding to the native structure. Analysis of ribosome intermediate structures indicates that this role for assembly factors is ubiquitous and suggests that helical junctions present significant obstacles to folding.


Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 738
Author(s):  
Jiayi Qian ◽  
Siyuan Su ◽  
Pengda Liu

The mTOR signaling controls essential biological functions including proliferation, growth, metabolism, autophagy, ageing, and others. Hyperactivation of mTOR signaling leads to a plethora of human disorders; thus, mTOR is an attractive drug target. The discovery of mTOR signaling started from isolation of rapamycin in 1975 and cloning of TOR genes in 1993. In the past 27 years, numerous research groups have contributed significantly to advancing our understanding of mTOR signaling and mTOR biology. Notably, a variety of experimental approaches have been employed in these studies to identify key mTOR pathway members that shape up the mTOR signaling we know today. Technique development drives mTOR research, while canonical biochemical and yeast genetics lay the foundation for mTOR studies. Here in this review, we summarize major experimental approaches used in the past in delineating mTOR signaling, including biochemical immunoprecipitation approaches, genetic approaches, immunofluorescence microscopic approaches, hypothesis-driven studies, protein sequence or motif search driven approaches, and bioinformatic approaches. We hope that revisiting these distinct types of experimental approaches will provide a blueprint for major techniques driving mTOR research. More importantly, we hope that thinking and reasonings behind these experimental designs will inspire future mTOR research as well as studies of other protein kinases beyond mTOR.


2020 ◽  
Vol 48 (14) ◽  
pp. 8128-8145 ◽  
Author(s):  
Katy Stokes ◽  
Alicja Winczura ◽  
Boyuan Song ◽  
Giacomo De Piccoli ◽  
Daniel B Grabarczyk

Abstract The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.


Sign in / Sign up

Export Citation Format

Share Document