Genome Sequencing and Comparative Analysis of Saccharomyces cerevisiae Strains of the Peterhof Genetic Collection

The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet. Here we employed whole genome sequencing to characterize five selected PGC strains including one of the closest to the progenitor, 15V-P4, and several strains that have been used to study translation termination and prions in yeast (25-25-2V-P3982, 1B-D1606, 74-D694, and 6P-33G-D373). The genetic distance between the PGC progenitor and S288C is comparable to that between two geographically isolated populations. The PGC seems to be closer to two bakery strains than to S288C-related laboratory stocks or European wine strains. In genomes of the PGC strains, we found several loci which are absent from the S288C genome; 15V-P4 harbors a rare combination of the gene cluster characteristic for wine strains and the RTM1 cluster. We closely examined known and previously uncharacterized gene variants of particular strains and were able to establish the molecular basis for known phenotypes including phenylalanine auxotrophy, clumping behavior and galactose utilization. Finally, we made sequencing data and results of the analysis available for the yeast community. Our data widen the knowledge about genetic variation between Saccharomyces cerevisiae strains and can form the basis for planning future work in PGC-related strains and with PGC-derived alleles.PBD acknowledges the Russian Foundation for Basic Research (www.rfbr.ru) for grant 14-04-31265. OVT and SGIV acknowledge the Russian Foundation for Basic Research for grant 15-29-02526. JVS acknowledges the Russian Science Foundation (www.rscf.ru) for grant 14-50-00069 and the Saint-Petersburg State University for grant 1.38.426.2015. PBD, AGM, EAR, and JVS acknowledge the Saint-Petersburg State University for research grant 1.37.291.2015. PBD and OVT acknowledge the Saint-Petersburg City Committee on Science and High School (knvsh.gov.spb.ru/) for grants 15404 and 15919, respectively. PBD, AGM, JVS, and SGIV acknowledge the Saint-Petersburg State University for research grant 15.61.2218.2013. PBD acknowledges the Saint-Petersburg State University for research grant 1.42.1394.2015

Search for Structural Basis of Interactions of Biogenic Amines with Human TAAR1 and TAAR6 Receptors

The identification and characterization of ligand-receptor binding sites are important for drug development. Trace amine-associated receptors (TAARs, members of the class A GPCR family) can interact with different biogenic amines and their metabolites, but the structural basis for their recognition by the TAARs is not well understood. In this work, we have revealed for the first time a group of conserved motifs (fingerprints) characterizing TAARs and studied the docking of aromatic (β-phenylethylamine, tyramine) and aliphatic (putrescine and cadaverine) ligands, including gamma-aminobutyric acid, with human TAAR1 and TAAR6 receptors. We have identified orthosteric binding sites for TAAR1 (Asp68, Asp102, Asp284) and TAAR6 (Asp78, Asp112, Asp202). By analyzing the binding results of 7500 structures, we determined that putrescine and cadaverine bind to TAAR1 at one site, Asp68 + Asp102, and to TAAR6 at two sites, Asp78 + Asp112 and Asp112 + Asp202. Tyramine binds to TAAR6 at the same two sites as putrescine and cadaverine and does not bind to TAAR1 at the selected Asp residues. β-Phenylethylamine and gamma-aminobutyric acid do not bind to the TAAR1 and TAAR6 receptors at the selected Asp residues. The search for ligands targeting allosteric and orthosteric sites of TAARs has excellent pharmaceutical potential

New PAM Improves the Single-Base Specificity of crRNA-Guided LbCas12a Nuclease

The RNA-guided Cas12a nuclease forms a complex with a CRISPR RNA (crRNA) to cleave the double-stranded DNA target. Among others, Cas12a protein from Lachnospiraceae bacterium (LbCas12a) is widely used for biomedical research. For target recognition, LbCas12a requires a specific nucleotide sequence, named a protospacer adjacent motif (PAM). Besides the canonical TTTV PAM, LbCas12a can recognize other suboptimal PAMs. We examined a novel TTAA PAM for the LbCas12a nuclease and found that the specificity of cleavage was increased. We found that single nucleotide substitutions at all positions of the guide RNA except the 20th position blocked the cleavage of the target DNA. The type of nucleotide substitutions (U-A, U-C or U-G) did not affect the efficiency of cleavage in the 20th position. When we used the canonical PAM under the same conditions, we observed the cleavage of target DNA by LbCas12a in many positions, showing less specificity in given conditions. The efficiency and specificity of the LbCas12a nuclease were evaluated both by gel-electrophoresis and using FAM-labeled single-stranded probes. We were able to assess the change in fluorescence intensity only for several variants of guide RNAs. High specificity allows us to type single nucleotide substitutions and small deletions/insertions (1–2 nucleotides) and look for target mutations when knocking out

Functional Mammalian Amyloids and Amyloid-Like Proteins

Amyloids are highly ordered fibrous cross-β protein aggregates that are notorious primarily because of association with a variety of incurable human and animal diseases (termed amyloidoses), including Alzheimer’s disease (AD), Parkinson’s disease (PD), type 2 diabetes (T2D), and prion diseases. Some amyloid-associated diseases, in particular T2D and AD, are widespread and affect hundreds of millions of people all over the world. However, recently it has become evident that many amyloids, termed “functional amyloids,” are involved in various activities that are beneficial to organisms. Functional amyloids were discovered in diverse taxa, ranging from bacteria to mammals. These amyloids are involved in vital biological functions such as long-term memory, storage of peptide hormones and scaffolding melanin polymerization in animals, substrate attachment, and biofilm formation in bacteria and fungi, etc. Thus, amyloids undoubtedly are playing important roles in biological and pathological processes. This review is focused on functional amyloids in mammals and summarizes approaches used for identifying new potentially amyloidogenic proteins and domains

Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae.

The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI+] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI+] strains is determined by the interaction between [SWI+] and [PIN+] prions. Using genetic and biochemical methods, we showed that [SWI+] is the key determinant of this nonsense suppression, whereas [PIN+] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI+]. We demonstrated that interaction of [SWI+] and [PIN+] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae

Specificities of Scanning Electron Microscopy and Histological Methods in Assessing Cell-Engineered Construct Effectiveness for the Recovery of Hyaline Cartilage

Damage to the hyaline layer of the articular surface is an urgent problem for millions of people around the world. At present, a large number of experimental methods are being developed to address this problem, including the transplantation of a cell-engineered construct (CEC) composed of a biodegradable scaffold with a premixed cell culture into the damaged area of the articular surface. However, current methods for analyzing the effectiveness of such CECs have significant limitations. This study aimed to compare the SEM technique, classical histology, and cryosectioning for the analysis of CECs transplanted to hyaline cartilage

Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. They modulate key physiological functions and are required in diverse developmental processes including embryogenesis, but their role in pluripotency maintenance and acquisition during the reprogramming towards hiPSCs draws little attention. Meanwhile, it is known that more than 106 GPCRs are overexpressed in human pluripotent stem cells (hPSCs). Previously, to identify novel effectors of reprogramming, we performed a high-throughput RNA interference (RNAi) screening assay and identified adhesion GPCR, GPR123, as a potential reprogramming effector. Its role has not been explored before. Herein, by employing GPR123 RNAi we addressed the role of GPR123 for hPSCs. The suppression of GPR123 in hPSCs leads to the loss of pluripotency and differentiation, impacted colony morphology, accumulation of cells at the G2 phase of the cell cycle, and absence of the scratch closure. Application of the GPR123 RNAi at the initiation stage of reprogramming leads to a decrease in the percentage of the “true” hiPSC colonies, a drop in E-cadherin expression, a decrease in the percentage of NANOG+ nuclei, and the absence of actin cytoskeleton remodeling. Together this leads to the absence of the alkaline-phosphatase-positive hiPSCs colonies on the 18th day of the reprogramming process. Overall, these data indicate for the first time the essential role of GPR123 in the maintenance and acquisition of pluripotency

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro

RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-β fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-β structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation

2D-DIGE image of proteins forming SDS-insoluble aggregates isolated from the 1-1-D931 [<i>NSI</i>⁺] and 1-1-1-D931 [<i>nsi</i>^-] strains.

<p>Spots corresponding to proteins from 1-1-D931 [<i>NSI</i>⁺] cells are pseudocolored in red (Cy5), while proteins from the 1-1-1-D931 [<i>nsi</i>^-] are pseudocolored in green (Cy3). Yellow spots correspond to proteins present in both samples. A strip with a pH gradient of 5–8 was used. Proteins identified by mass-spectrometry are indicated. The mass spectra of identified proteins are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006504#pgen.1006504.s001" target="_blank">S1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006504#pgen.1006504.s003" target="_blank">S3</a> Figs.</p

Genes absent from the S288C genome but found in 15V-P4.

<p>Genes absent from the S288C genome but found in 15V-P4.</p