scholarly journals High-throughput functional analysis of natural variants in yeast

2021 ◽  
Author(s):  
Chiann-Ling C Yeh ◽  
Andreas Tsouris ◽  
Joseph Schacherer ◽  
Maitreya J. Dunham
Keyword(s):  
High Throughput ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Evolutionary Pattern ◽  
Intermediate Phenotypes ◽  
Sulfate Limitation ◽  
Long Read ◽  
Natural Variants ◽  
Natural Isolates ◽  
Error Correction Algorithm

How natural variation affects phenotype is difficult to determine given our incomplete ability to deduce the functional impact of the polymorphisms detected in a population. Although current computational and experimental tools can predict and measure allele function, there has previously been no assay that does so in a high-throughput manner while also representing haplotypes derived from wild populations. Here, we present such an assay that measures the fitness of hundreds of natural alleles of a given gene without site-directed mutagenesis or DNA synthesis. With a large collection of diverse Saccharomyces cerevisiae natural isolates, we piloted this technique using the gene SUL1, which encodes a high-affinity sulfate permease that, at increased copy number, can improve the fitness of cells grown in sulfate-limited media. We cloned and barcoded all alleles from a collection of over 1000 natural isolates en masse and matched barcodes with their respective variants using PacBio long-read sequencing and a novel error-correction algorithm. We then transformed the reference S288C strain with this library and used barcode sequencing to track growth ability in sulfate limitation of lineages carrying each allele. We show that this approach allows us to measure the fitness conferred by each allele and stratify functional and nonfunctional alleles. Additionally, we pinpoint which polymorphisms in both coding and noncoding regions are detrimental to fitness or are of small effect and result in intermediate phenotypes. Integrating these results with a phylogenetic tree, we observe how often loss-of-function occurs and whether or not there is an evolutionary pattern to our observable phenotypic results. This approach is easily applicable to other genes. Our results complement classic genotype-phenotype mapping strategies and demonstrate a high-throughput approach for understanding the effects of polymorphisms across an entire species which can greatly propel future investigations into quantitative traits.

scholarly journals Color-tuning of natural variants of heliorhodopsin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Se-Hwan Kim ◽  
Kimleng Chuon ◽  
Shin-Gyu Cho ◽  
Ahreum Choi ◽  
Seanghun Meas ◽  
...  
Keyword(s):  
3D Structure ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Transmembrane Helices ◽  
Color Tuning ◽  
Tuning Mechanism ◽  
Spectral Changes ◽  
His Tag ◽  
Natural Variants ◽  
Retinal Chromophore

AbstractMicrobial rhodopsins are distributed through many microorganisms. Heliorhodopsins are newly discovered but have an unclear function. They have seven transmembrane helices similar to type-I and type-II rhodopsins, but they are different in that the N-terminal region of heliorhodopsin is cytoplasmic. We chose 13 representative heliorhodopsins from various microorganisms, expressed and purified with an N-terminal His tag, and measured the absorption spectra. The 13 natural variants had an absorption maximum (λmax) in the range 530–556 nm similar to proteorhodopsin (λmax = 490–525 nm). We selected several candidate residues that influence rhodopsin color-tuning based on sequence alignment and constructed mutants via site-directed mutagenesis to confirm the spectral changes. We found two important residues located near retinal chromophore that influence λmax. We also predict the 3D structure via homology-modeling of Thermoplasmatales heliorhodopsin. The results indicate that the color-tuning mechanism of type-I rhodopsin can be applied to understand the color-tuning of heliorhodopsin.

Chloroethyl urea derivatives block tumour growth and thioredoxin-1 nuclear translocation

2010 ◽  
Vol 88 (11) ◽  
pp. 1102-1114 ◽  
Author(s):  
Alexandre Patenaude ◽  
Jessica S. Fortin ◽  
Réna Deschenes ◽  
Marie-France Côté ◽  
Jacques Lacroix ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Nuclear Translocation ◽  
Human Cancer ◽  
Alkylating Agents ◽  
Covalent Binding ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Thioredoxin 1

Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that 14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.

scholarly journals The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6806
Author(s):  
Bruna F. Mazzeu ◽  
Tatiana M. Souza-Moreira ◽  
Andrew A. Oliveira ◽  
Melissa Remlinger ◽  
Lidiane G. Felippe ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activities ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enzyme Specificity ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Pentacyclic Triterpene ◽  
Oxidosqualene Cyclases ◽  
Maytenus Ilicifolia

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

scholarly journals Oxford Nanopore Sequencing, Hybrid Error Correction, and de novo Assembly of a Eukaryotic Genome

2015 ◽  
Author(s):  
Sara Goodwin ◽  
James Gurtowski ◽  
Scott Ethe-Sayers ◽  
Panchajanya Deshpande ◽  
Michael Schatz ◽  
...  
Keyword(s):  
Error Correction ◽  
De Novo Assembly ◽  
De Novo ◽  
Correction Algorithm ◽  
Membrane Pore ◽  
Complete Representation ◽  
Oxford Nanopore ◽  
Long Read ◽  
Error Correction Algorithm ◽  
Sequencing Instrument

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available that we used for sequencing the S. cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr (https://github.com/jgurtowski/nanocorr) specifically for Oxford Nanopore reads, as existing packages were incapable of assembling the long read lengths (5-50kbp) at such high error rate (between ~5 and 40% error). With this new method we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: the contig N50 length is more than ten-times greater than an Illumina-only assembly (678kb versus 59.9kbp), and has greater than 99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.

scholarly journals Influence of Ferroportin Disease Mutations on Manganese Accumulation and Toxicity (P24-060-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Eun-kyung Choi ◽  
Young-Ah Seo
Keyword(s):  
Genetic Disorder ◽  
Functional Results ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Excess Iron ◽  
Funding Sources ◽  
Disease Mutations ◽  
The Impact

Abstract Objectives Hemochromatosis is a frequent genetic disorder characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell-surface iron exporter ferroportin (Fpn), are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill defined, and the impact of disease mutations on cellular Mn levels is unknown. Thus, this study aimed to define the role of Fpn in Mn regulation and determine the functional consequences of ferroportin disease mutations in cellular Mn levels. Methods Thus far, over 50 mutations in Fpn have been identified in hemochromatosis type 4/ferroportin disease. To test whether these mutations alter cellular Mn metabolism, we constructed an expression vector encoding human Fpn with a C-terminal HA epitope tag and introduced nine clinically relevant mutations by site-directed mutagenesis. Based on previously reported in vitro functional results, we selected five ferroportin disease mutations from each of the two groups: five loss-of-function (LOF) mutations (G80S, R88G, D157G, D157Y, and V162Δ) and four gain-of-function (GOF) mutations (N144H, N144T, C326S, and and S338R). Results Here, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn appears to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with Fpn's role in controlling Mn levels as well as the stability of Fpn. Conclusions These results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease. Funding Sources National Institutes of Health (NIH) to Y.A.S. (K99/R00 ES024340).

scholarly journals Avian MHC Evolution in the Era of Genomics: Phase 1.0

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1152 ◽  
Author(s):  
Emily A. O’Connor ◽  
Helena Westerdahl ◽  
Reto Burri ◽  
Scott V. Edwards
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Structural Evolution ◽  
Great Depth ◽  
Long Read ◽  
Ecological Patterns ◽  
In The Wild ◽  
The Many ◽  
Avian Mhc

Birds are a wonderfully diverse and accessible clade with an exceptional range of ecologies and behaviors, making the study of the avian major histocompatibility complex (MHC) of great interest. In the last 20 years, particularly with the advent of high-throughput sequencing, the avian MHC has been explored in great depth in several dimensions: its ability to explain ecological patterns in nature, such as mating preferences; its correlation with parasite resistance; and its structural evolution across the avian tree of life. Here, we review the latest pulse of avian MHC studies spurred by high-throughput sequencing. Despite high-throughput approaches to MHC studies, substantial areas remain in need of improvement with regard to our understanding of MHC structure, diversity, and evolution. Recent studies of the avian MHC have nonetheless revealed intriguing connections between MHC structure and life history traits, and highlight the advantages of long-term ecological studies for understanding the patterns of MHC variation in the wild. Given the exceptional diversity of birds, their accessibility, and the ease of sequencing their genomes, studies of avian MHC promise to improve our understanding of the many dimensions and consequences of MHC variation in nature. However, significant improvements in assembling complete MHC regions with long-read sequencing will be required for truly transformative studies.

Using PacBio Long-Read High-Throughput Microbial Gene Amplicon Sequencing To Evaluate Infant Formula Safety

2016 ◽  
Vol 64 (37) ◽  
pp. 6993-7001 ◽  
Author(s):  
Yi Zheng ◽  
Xiaoxia Xi ◽  
Haiyan Xu ◽  
Qiangchuan Hou ◽  
Yanfei Bian ◽  
...  
Keyword(s):  
Infant Formula ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Long Read

scholarly journals MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

2018 ◽  
Vol 49 (1) ◽  
pp. 144-159 ◽  
Author(s):  
Ye Yuan ◽  
Fubiao Niu ◽  
Ilja M. Nolte ◽  
Jasper Koerts ◽  
Debora de Jong ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Western Blotting ◽  
High Throughput ◽  
Annexin V ◽  
Loss Of Function ◽  
Empty Vector ◽  
Competition Assays

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

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