Biology subjects

Sun, G.

Publications and source records attributed to Sun, G..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Population level gene expression can repeatedly link genes to functions in maize

Transcriptome-Wide Association Studies (TWAS) can provide single gene resolution for candidate genes in plants, complementing Genome-Wide Association Studies (GWAS) but efforts in plants have been met with, at best, mixed success. We generated expression data from 693 maize genotypes, measured in a common field experiment, sampled over a two-hour period to minimize diurnal and environmental effects, using full-length RNA-seq to maximize the accurate estimation of transcript abundance. TWAS could identify roughly ten times as many genes likely to play a role in flowering time regulation as GWAS conducted data from the same experiment. TWAS using mature leaf tissue identified known true positive flowering time genes known to act in the shoot apical meristem, and trait data from new environments enabled the identification of additional flowering time genes without the need for new expression data. eQTL analysis of TWAS-tagged genes identified at least one additional known maize flowering time gene through trans-eQTL interactions. Collectively these results suggest the gene expression resource described here can link genes to functions across different plant phenotypes expressed in a range of tissues and scored in different experiments.

plant biology↗

bioRxiv2023

Identification and characterization of a beta-1,4 Galactosidase from Elizabethkingia meningoseptica and its application on living cell surface

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37) and pH (5-8) levels and can remove {beta}-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As the enzyme has a potential application for living cell glycan editing, we named it glycan editing galactosidase I or geGalaseI. In summary, our findings lay the groundwork for prospective investigations by presenting a prompt and gentle approach for the removal of galactose moieties from cell surface.

biochemistry↗

bioRxiv2023

The apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favour and against this idea. RNase treatment of chromatin is a prevalent tool for the study of RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (RNase-ChIP or rChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase PRC2. This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here we show that RNase A treatment during chromatin immunoprecipitation leads to the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome wide. This phenomenon persists in mouse embryonic stem cells, human cancer cells and human-induced pluripotent stem cells. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results point to substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay. HighlightsO_LIRNA degradation during ChIP-seq is insufficient to displace PRC2 from chromatin. C_LIO_LIRNA degradation led to the artificial depletion of ChIP-seq signals in multiple cell lines. C_LIO_LIArtificially reduced ChIP-seq signals are explained by a gain of non-targeted DNA. C_LIO_LIRNA is critical in maintaining the solubility of chromatin during experimentation. C_LI

molecular biology↗

bioRxiv2023

Phenotypic variation in maize can be largely explained by genetic variation at transcription factor binding sites

Comprehensive maps of functional variation at transcription factor (TF) binding sites (cis-elements) are crucial for elucidating how genotype shapes phenotype. Here we report the construction of a pan-cistrome of the maize leaf under well-watered and drought conditions. We quantified haplotype-specific TF footprints across a pan-genome of 25 maize hybrids and mapped over two-hundred thousand genetic variants (termed binding-QTL) linked to cis-element occupancy. Three lines of evidence support the functional significance of binding-QTL: i) they coincide with numerous known causative loci that regulate traits, including VGT1, Trehalase1, and the MITE transposon near ZmNAC111 under drought; ii) their footprint bias is mirrored between inbred parents and by ChIP-seq; iii) partitioning genetic variation across genomic regions demonstrates that binding-QTL capture the majority of heritable trait variation across [~]70% of 143 phenotypes. Our study provides a promising approach to make previously hidden cis-variation more accessible for genetic studies and multi-target engineering of complex traits.

genomics↗

bioRxiv2023

Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Fast and accurate DNASeq Variant Calling workflow composed of LUSH toolkit

BackgroundWhole genome sequencing (WGS) is becoming increasingly prevalent for molecular diagnosis, staging and prognosis because of its declining costs and the ability to detect nearly all genes associated with a patients disease. The currently widely accepted variant calling pipeline, GATK, is limited in terms of its computational speed and efficiency, which cannot meet the growing analysis needs. MethodsIn this study, we propose a fast and accurate DNASeq variant calling workflow that is purely composed of tools from LUSH toolkit. The LUSH pipeline is highly optimized for the WGS pipeline based on SOAPnuke, BWA and GATK which can be deployed on any general-purpose CPU-based computing system. We validated the accuracy, speed and scalability of the LUSH pipeline on several standard WGS datasets. ResultsOur test results show that the LUSH pipeline and the GATK pipeline are highly consistent in terms of accuracy, achieving over 99% precision and recall on NA12878. For speed, the LUSH pipeline completes 30x WGS data in 1.6 hours, which is about 17x faster than the GATK pipeline. From BAM to VCF, LUSH_HC even takes only 12 minutes, about 76x faster than GATK. Moreover, the LUSH pipeline shows favorable scalability in terms of thread and sequencing depth. ConclusionThe LUSH pipeline provides far superior computational speed to GATK while maintaining a high level of accuracy comparable to that of GATK, which greatly facilitates bedside analysis of acute patients, large-scale cohort data analysis, and variant calling in crop breeding programs.

bioinformatics↗

bioRxiv2023

Duck pan-genome reveals two transposon-derived structural variations caused bodyweight enlarging and white plumage phenotype formation during evolution

Structural variations (SVs) are a major source of domestication and improvement traits, however SV profiles of duck and their phenotypic impacts largely hidden. We present the first duck pan-genome constructed using five genome assemblies capturing [~]40.98 Mb new sequences. This pan-genome together with high-depth sequencing data ([~]46.5X) identified 101,041 SVs, of which substantial proportions were derived from transposable element (TE) activity. Many TE-derived SVs anchoring in a gene body or regulatory region are linked to ducks domestication and improvement. By combining quantitative genetics with molecular experiments, we dissect how TE-derived SVs change gene expression of IGF2BP1 and generate a novel transcript of MITF, shaping bodyweight and white plumage. In the IGF2BP1 locus, the TE-derived SV explains the largest effect on bodyweight among avian species (27.61% of phenotypic variation). Our findings highlight the importance of using a pan-genome as a reference in genomics studies and explore the roles of TE-derived SVs in trait formation and in livestock breeding.

genetics↗

bioRxiv2022

MYSM1 co-activates ERα action via histone and non-histone deubiquitination to confer antiestrogen resistance in breast cancer

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ER)-positive breast cancer (BCa) therapy. Aberrant alteration in modulation of E2/ER signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Thus, identification the efficient ER cofactor remains necessary for finding a potential therapeutic target for endocrine resistance. Herein, we have demonstrated that Myb like, SWIRM and MPN domains 1 (MYSM1) as a histone deubiquitinase is a novel ER co-activator with established Drosophila experimental model. Our results showed that MYSM1 participated in up-regulation of ER action via histone and non-histone deubiquitination. We provided the evidence to show that MYSM1 was involved in maintenance of ER stability via ER deubiquitination. Furthermore, silencing MYSM1 induced enhancement of histone H2A ubiquitination as well as reduction of histone H3K4me3 and H3Ac levels at cis regulatory elements on promoter of ER-regulated gene. In addition, MYSM1 depletion attenuated cell proliferation/growth in BCa-derived cell lines and xenograft models. Knockdown of MYSM1 increased the sensitivity of antiestrogen agents in BCa cells. MYSM1 was highly expressed in clinical BCa samples, especially in aromatase inhibitor (AI) non-responsive tissues. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ER action and provide a potential therapeutic target for endocrine resistance in BCa.

cell biology↗

bioRxiv2022

Measurement of expression from a limited number of genes is sufficient to predict flowering time in maize

Changing patterns of weather and climate are limiting breeders ability to conduct trials in the same environments in which their released varieties will be grown 7-10 years later. Flowering time plays a crucial role in determining regional adaptation, and mismatch between flowering time and environment can substantially impair yield. Different approaches based on genetic markers or gene expression can be used to predict flowering time before conducting large scale field evaluation and phenotyping. The more accurate prediction of a trait using genetic markers could be hindered due to all the intermediate steps (i.e. transcription, translation, epigenetic modification, and epistasis among others) connecting the trait and their genetic basics. The use of some intermediate steps as predictors could improve the accuracy of the model. Here, we are using two public gene expression (RNA-Seq) data-sets from 14-day-old-maize-seedling roots and whole-seedling tissue at v1 stage (10 day after planting) for which flowering data (days to anthesis and days to silking expressed in growing degree days) and genetic markers were also available to test the predictability of flowering time. In total, 20 different combinations between phenotypic and gene expression data-sets were evaluated. To explore prediction accuracy a random forest model was trained with the expression values of 44,303 gene models hosted in the current B73 maize reference version 5 and then the feature importance was scored based on the decrease in root mean squared error. Later several random forest models with different subsets of the most important features (genes) were trained, and this process was repeated ten times. Results from these analyses show a curve in the prediction accuracy, with an increase in the prediction accuracy as the top most important genes were added. The maximum accuracy was attained when 500 genes for whole-seedling and 100 genes for root gene expression data were used in the analysis, and thereafter adding more genes lead to a decrease in the prediction accuracy. The highest prediction accuracy using the top-most important genes was higher than that of using randomly selected whole-genome 400,000 SNPs. Finally, we described the genes controlling flowering time by looking at the most important genes in the Random forest model with the expression data from all genes. We further found MADS-transcription factor 69 (Mads69) using whole-seedling gene expression and the MADS-transcription factor 67 (Mads67) using root gene expression data, both genes previously described with effect on flowering time. Here, we aim to demonstrate the potential of selecting and using the expression of most informative genes to predict a complex trait, also to demonstrate the robustness and limitations of this analysis by using phenotypic data-sets from different environments.

genomics↗

bioRxiv2022

Innate immune response to SARS-1 CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons

Severe acute respiratory coronavirus 2 (SARS-CoV-2) infection causes neurological disease in some patients suggesting that infection can affect both the peripheral and central nervous system (PNS and CNS, respectively). It is not clear whether the outcome of SARS-CoV-2 infection of PNS and CNS neurons is similar, and which are the key factors that cause neurological disease: SARS-CoV-2 infection or the subsequent immune response. Here, we addressed these questions by infecting human induced-pluripotent stem cell-derived CNS and PNS neurons with the {beta} strain of SARS-CoV-2. Our results show that SARS-CoV-2 infects PNS neurons more efficiently than CNS neurons, despite lower expression levels of angiotensin converting enzyme 2. Infected PNS neurons produced interferon {lambda}1, several interferon stimulated genes and proinflammatory cytokines. They also displayed neurodegenerative-like alterations, as indicated by increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and -synuclein and lower levels of nicotinamide mononucleotide adenylyltransferase 2 and {beta}-III-tubulin. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neurodegeneration, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS.

microbiology↗

bioRxiv2022

Parallel use of pluripotent human stem cell lung and heart models provide new insights for treatment of SARS-CoV-2

SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems. One-sentence summaryRational treatment strategies for SARS-CoV-2 derived from human PSC models

microbiology↗

bioRxiv2022

Reassessing the abundance of miRNAs in the human pancreas and rodent cell lines and its implication

miRNAs are critical for pancreas development and function. However, we found that there are discrepancies of pancreatic miRNA abundance in published datasets. To obtain a more relevant profile that is close to the true profile, we profiled small RNAs from human islets cells, acini, and four rodent pancreatic cell lines routinely used in diabetes and pancreatic research using a bias reduction protocol for small RNA sequencing. In contrast to the previous notion that miR-375-3p is the sole dominant pancreatic miRNA, we found that miR-148a-3p and miR-7-5p were equally abundant in islets. In silico studies using predicted and validated targets of these three miRNAs revealed that they possibly work in cooperation to modulate cell differentiation in endocrine and exocrine cells. Our results also suggest, compare to the well characterized miR-375, both miR-148a-3p and miR-7-5p may play more critical roles in human pancreas development and function. Moreover, according to in silico predicted targets, we found that miR-375-3p had a much broader target spectrum by targeting the coding sequence and the 5 untranslated region rather than the conventional 3 untranslated region, suggesting additional unexplored roles for miR-375-3p beyond pancreas. Our study provides a valuable resource for studying miRNAs in pancreas.

molecular biology↗

bioRxiv2022

Activation of Keap1 and its isoforms (α and β) inhibits the Nrf2 pathway to affect malignant behaviour of human hepatocellular carcinoma cells

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification and cytoprotective responses against oxidative, electrophilic and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and hence changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 and {beta} have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1-/-, Keap1{beta} (Keap1{Delta}1-31), Keap1-Restored and Keap1-Restored, aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furtherly, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e. subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity and intracellular GSH levels). Of note, the results obtained from tumorigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial-mesenchymal transition), cell cycle and apoptosis.

biochemistry↗

bioRxiv2022

Distinct roles of Nrf1 and Nrf2 in monitoring the reductive stress response to dithiothreitol (DTT)

Transcription factor Nrf2 (nuclear factor, erythroid 2-like 2, encoded by Nfe2l2) has been accepted as a key player in redox regulatory responses to oxidative or reductive stresses. However, it is less or not known about the potential role for Nrf1 (nuclear factor, erythroid 2-like 1, encoded by Nfe2l1) in the redox responses, particularly to reductive stress, albeit this fossil-like factor is indispensable for cell homeostasis and organ integrity during life process. Here, we examine distinct roles of Nrf1 and Nrf2 in monitoring the defense response to 1,4-dithiothreitol (DTT, serving as a reductive stressor), concomitantly with unfolded protein response being induced by this chemical (also as an endoplasmic reticulum stressor). The results revealed that intracellular reactive oxygen species (ROS) were modestly increased in DTT-treated wild-type (WT) and Nrf1-/- cell lines, but almost unaltered in Nrf2-/-{Delta}TA or caNrf2{Delta}N cell lines (with a genetic loss of its transactivation or N-terminal Keap1-binding domains, respectively). This chemical treatment also enabled the rate of oxidized to reduced glutathione (i.e., GSSG to GSH) to be amplified in WT and Nrf2-/-{Delta}TA cells, but diminished in Nrf1-/- cells, along with no changes in caNrf2{Delta}N cells. Consequently, Nrf1-/-, but not Nrf2-/-{Delta}TA or caNrf2{Delta}N, cell viability was reinforced by DTT against its cytotoxicity, as accompanied by decreased apoptosis. Further experiments unraveled that Nrf1 and Nrf2 differentially, and also synergistically, regulated DTT-inducible expression of critical genes for defending redox stress and endoplasmic reticulum stress. In addition, we have also identified that Cys342 and Cys640 of Nrf1 (as redox-sensing sites within its N-glycodomain and DNA-binding domain, respectively) are required for its protein stability and transcription activity.

cell biology↗

bioRxiv2022

Comparative genomics of five Valsa species gives insights on their pathogenicity evolution

Valsa is a genus of ascomycetes fungi within the family Valsaceae that includes many wood destructive pathogens. The species such as Valsa mali and Valsa pyri that colonize fruit trees are threatening the global fruit production. Rapid host adaptation and fungicide resistance emergence are the main characteristics that make them devastating and hard to control. Efficient disease management can be achieved from early infection diagnosis and fungicide application, but lack of understandings of their genetic diversity and genomic features that underpin their pathogenicity evolution and drug resistance is essentially impeding the progress of effective and sustainable disease control. Here, we report genome assemblies of Valsa malicola, Valsa persoonii and Valsa sordida which represents close relatives of the two well known Valsa mali and Valsa pyri that cause canker disease with different host preferences. Comparative genomics analysis revealed that segmental rearrangements, inversions and translocations frequently occurred among Valsa spp. genomes. Genes identified in highly active regions exhibited high sequence differentiation and are enriched in membrane transporter proteins involved in anti-drug and nutrient transportation activities. Consistently, we also found membrane transporter gene families have been undergoing significant expansions in Valsa clade. Furthermore, unique genes that possessed or retained by each of the five Valsa species are more likely part of the secondary metabolic (SM) gene clusters which suggests SM one of the critical components that diverge along with the evolution of Valsa species. Repeat sequence content contributes significantly to genome size variation across the five species. The wide spread AT-rich regions resulted from repeat induced point C to T mutation (RIP) exhibited a specific proximity to secondary metabolic gene clusters and this positional proximity is correlated with the diversification of SM clusters suggesting a potential companion evolution between repeat sequence and secondary metabolism cluster. Lastly, we show that LaeA, the global regulator of secondary metabolic gene cluster, exhibiting diverged manner of regulation on the expression of clusters in vegetative and invasive mycelia of the devastating V. mali indicating the complexity of secondary metabolism in fungal species.

bioinformatics↗

bioRxiv2022

An Efficient CRISPR-Cas9 DNA Editing Methodology Applicable for iPSC Disease Modelling

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularised CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labour-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This was accomplished through improved viability of the transfected cells, paralleled with provision of enriched environment for the robust establishment and proliferation of singularised iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50-65% for NHEJ and 10% for HDR, indicative of the methods utility in stem cell disease modelling.

genetics↗

bioRxiv2022

A full spectrum PNGase activity analysis of R328 mutations on NGLY1

In humans, N-glycanase 1 (NGLY1; Peptide: N-glycanase, PNGase) is responsible for the deglycosylation of misfolded glycoproteins. Pathogenic mutations in NGLY1 cause a clinical condition known as congenital disorder of deglycosylation (NGLY1-CDDG), a rare autosomal recessive disease first reported in 2012. Although NGLY1-CDDG was diagnosed through whole-exome or whole-genome sequencing and by evaluating the expression levels of NGLY1, the clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, an in vitro enzymatic assay system was established to evaluate the thermal stability and substrate specificity of NGLY1, as well as the optimum reaction conditions for its activity. A panel of all mutations at the amino acid site R328 in NGLY1 was subjected to this assay. The results revealed that R328A, R328D, R328E, R328F, R328G, R328I, R328P, R328V, R328W, and R328Y were dysfunctional mutations (10/19); NGLY1 mutations with R328H and R328T exhibited similar activity as wild-type NGLY1 (2/19); and NGLY1 mutations with R328C, R328K, R328L, R328M, R328N, R328Q, and R328S showed decreased activity (7/19) compared to wild-type NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-L-cysteine and dithiothreitol, on NGLY1 was examined. This in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations on NGLY1 and a practical screening method for drugs and compounds with potential therapeutic value for NGLY1-CDDG patients.

biochemistry↗

bioRxiv2022

Association Mapping Across a Multitude of Traits Collected in Diverse Environments Identifies Pleiotropic Loci in Maize.

Classical genetic studies have identified many cases of pleiotropy where mutations in individual genes alter many different phenotypes. Quantitative genetic studies of natural genetic variants frequently examine one or a few traits, limiting their potential to identify pleiotropic effects of natural genetic variants. Widely adopted community association panels have been employed by plant genetics communities to study the genetic basis of naturally occurring phenotypic variation in a wide range of traits. High-density genetic marker data - 18M markers - from two partially overlapping maize association panels comprising 1,014 unique genotypes grown in field trials across at least seven US states and scored for 162 distinct trait datasets enabled the identification of of 2,154 suggestive marker-trait associations and 697 confident associations in the maize genome using a resampling-based genome-wide association strategy. The precision of individual marker-trait associations was estimated to be three genes based a reference set of genes with known phenotypes. Examples were observed of both genetic loci associated with variation in diverse traits (e.g. above-ground and below-ground traits), as well as individual loci associated with the same or similar traits across diverse environments. Many significant signals are located near genes whose functions were previously entirely unknown or estimated purely via functional data on homologs. This study demonstrates the potential of mining community association panel data using new higher density genetic marker sets combined with resampling-based genome-wide association tests to develop testable hypotheses about gene functions, identify potential pleiotropic effects of natural genetic variants, and study genotype by environment interaction.

plant biology↗