Biology subjects

Zhao, R.

Publications and source records attributed to Zhao, R..

At least 163 records · Page 9Linked to original sources

bioRxiv2020

Optoregulated force application to cellular receptors using molecular motors

Mechanotransduction events in physiological environments are difficult to investigate, in part due to the lack of experimental tools to apply forces to mechanosensitive receptors remotely. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), here we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the polymer chains, which will in turn effectively "pulls" on engaged cell membrane receptors (integrins, cadherins...) within the illuminated area. Applied forces have the adequate magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in forcedependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate, for the first time, a functionality which at the moment cannot be achieved by any other means.

cell biology↗

bioRxiv2020

Fibrinolytic niche is requested for alveolar type 2 cell-mediated alveologenesis and injury repair

COVID-19, SARS, and MERS are featured by fibrinolytic dysfunction. To test the role of the fibrinolytic niche in the regeneration of alveolar epithelium, we compared the self-renewing capacity of alveolar epithelial type 2 (AT2) cells and its differentiation to AT1 cells between wild type (wt) and fibrinolytic niche deficient mice (Plau-/- and Serpine1Tg). A significant reduction in both proliferation and differentiation of deficient AT2 cells was observed in vivo and in 3D organoid cultures. This decrease was mainly restored by uPA derived A6 peptide, a binding fragment to CD44 receptors. The proliferative and differential rate of CD44+ AT2 cells was greater than that of CD44- controls. There was a reduction in transepithelial ion transport in deficient monolayers compared to wt cells. Moreover, we found a marked suppression in total AT2 cells and CD44+ subpopulation in lungs from brain dead patients with acute respiratory distress syndrome (ARDS) and a mouse model infected by influenza viruses. Thus, we demonstrate that the fibrinolytic niche can regulate AT2-mediated homeostasis and regeneration via a novel uPA-A6-CD44+-ENaC cascade.

cell biology↗

bioRxiv2020

Ion transport mechanisms for smoke inhalation injured airway epithelial barrier

Smoke inhalation injury is the leading cause of death in firefighters and victims. Inhaled hot air and toxic smoke are the predominant hazards to the respiratory epithelium. We aimed to analyze the effects of thermal stress and smoke aldehyde on the permeability of the airway epithelial barrier. Transepithelial resistance (RTE) and short-circuit current (ISC) of mouse tracheal epithelial monolayers were digitized by an Ussing chamber setup. Zonula occludens-1 tight junctions were visualized under confocal microscopy. A cell viability test and fluorescein isothiocyanate-dextran assay were performed. Thermal stress (40°C) decreased RTE in a two-phase manner. Meanwhile, thermal stress increased ISC followed by its decline. Na+ depletion, amiloride (an inhibitor for epithelial Na+ channels [ENaCs]), ouabain (a blocker for Na+/K+-ATPase) and CFTRinh-172 (a blocker of cystic fibrosis transmembrane regulator [CFTR]) altered the responses of RTE and ISC to thermal stress. Steady-state 40°C increased activity of ENaCs, Na+/K+-ATPase, and CFTR. Acrolein, one of the main oxidative unsaturated aldehydes in fire smoke, eliminated RTE and ISC. Na+ depletion, amiloride, ouabain, and CFTRinh-172 suppressed acrolein-sensitive ISC, but showed activating effects on acrolein-sensitive RTE. Thermal stress or acrolein disrupted zonula occludens-1 tight junctions, increased fluorescein isothiocyanate-dextran permeability but did not cause cell death or detachment. The synergistic effects of thermal stress and acrolein exacerbated the damage to monolayers. In conclusion, the paracellular pathway mediated by the tight junctions and the transcellular pathway mediated by active and passive ion transport pathways contribute to impairment of the airway epithelial barrier caused by thermal stress and acrolein.Graphical HeadlightsThermal stress and acrolein are two essential determinants for smoke-inhalation injury, impairing airway epithelial barrier.Transcellular ion transport pathways via the ENaC, CFTR, and Na/K-ATPase are interrupted by both thermal stress and acrolein, one of the most potent smoke toxins.Heat and acrolein damage the integrity of the airway epithelium through suppressing and relocating the tight junctions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsENaCsepithelial Na+ channelsCFTRcystic fibrosis transmembrane regulatorRTEtransepithelial resistanceISCshort-circuit currentMTEmouse tracheal epithelialFITCfluorescein isothiocyanateHBEhuman bronchial epithelialDMSOdimethyl sulfoxideZO-1zonula occludens-1P1phase 1P2phase 2ASIamiloride-sensitive ISCKCsK+ channelsROSreactive oxygen speciesCaCCsCa2+-activated Cl− channelsNKCCNa+/K+/2Cl−MAPKmitogen-activated protein kinaseView Full Text

pharmacology and toxicology↗

bioRxiv2020

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Background and PurposeLung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid. Experimental ApproachesHuman lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits. Key ResultsPlasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated {beta}{gamma}ENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts ({Delta}432-444, {gamma}{Delta}131-138, {gamma}{Delta}178-193, and {gamma}{Delta}410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human {gamma}ENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both and {gamma} subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Conclusion and ImplicationsWe demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases. Bullet point summaryO_ST_ABSWhat is already knowC_ST_ABSO_LISerine proteases proteolytically cleave epithelial sodium channels, including plasmin and uPA acutely. C_LIO_LIActivity of epithelial sodium channels is increased post proteolysis. C_LI What this study addsO_LIPlasmin cleaves up to 16 sites composed of two proteolytic centres in both full-length and furin-cleaved human {gamma} subunit of epithelial sodium channels in hours. C_LIO_LINon-proteolytic sites in both and {gamma} subunits interrupt the plasmin cleavage-induced channel gating. C_LIO_LIIntratracheally instilled plasmin facilitates alveolar fluid clearance in normal human and injured mouse lungs. C_LI Clinical significanceO_LIActivation of human lung epithelial sodium channels by plasmin may benefit lung oedema resolution as a novel therapy for ARDS. C_LI

pharmacology and toxicology↗

bioRxiv2020

Migration of Cytotoxic T Lymphocytes in 3D Collagen Matrices

CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main cytotoxic killer cells of the human body to eliminate pathogen-infected or tumorigenic cells (= target cells). To find their targets they have to navigate and migrate through a complex biological microenvironments, a key component of which is the extracellular matrix (ECM). The mechanisms underlying killer cells navigation are not well understood. To mimic an ECM we use a matrix formed by different collagen concentrations, and analyze migration trajectories of primary human CTLs. Different migration patterns are observed and can be grouped into three motility types: slow, fast and mixed. The dynamics are well described by a two-state persistent random walk model which allows cells to switch between slow motion with low persistence, and fast motion with high persistence. We hypothesize that the slow motility mode describes CTLs creating channels through the collagen matrix by deforming and tearing apart collagen fibers, and that the fast motility mode describes CTLs moving within these channels. Experimental evidence supporting this scenario is presented by visualizing migrating T cells following each other on exactly the same track and showing cells moving quickly in channel-like cavities within the surrounding collagen matrix. Consequently, the efficiency of the stochastic search process of CTLs in the ECM should strongly be influenced by a dynamically changing channel network produced by the killer cells themselves.

biophysics↗

bioRxiv2019

Cell type- and stage-specific expression of Otx2 is coordinated by a cohort of transcription factors and multiple cis-regulatory modules in the retina

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is an advantageous strategy. However, the mechanisms that control the activation of TFs in different cell types and at different stages of development remain unclear. The neural retina serves as a model of the development of a complex tissue. We used this system to analyze how expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during retinogenesis. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. These include mature bipolar interneurons, photoreceptors, and retinal progenitor/precursor cells. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages, as revealed by ATAC-seq and DNase-seq analyses. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2 in a cell type- and stage-specific manner in the retina.

developmental biology↗

bioRxiv2019

C-terminal binding proteins 1 and 2 in traumatic brain injury-induced inflammation and their inhibition as an approach for anti-inflammatory treatment

Traumatic brain injury (TBI) induces an acute inflammatory response in the central nervous system that involves both resident and peripheral immune cells. The ensuing chronic neuroinflammation causes cell death and tissue damage and may contribute to neurodegeneration. The molecular mechanisms involved in the maintenance of this chronic inflammation state remain underexplored. C-terminal binding protein (CtBP) 1 and 2 are transcriptional coregulators that repress diverse cellular processes. Unexpectedly, we find that the CtBPs can transactivate a common set of proinflammatory genes both in lipopolysaccharide-activated microglia, astrocytes and macrophages, and in a mouse model of the mild form of TBI. We also find that the expression of these genes is markedly enhanced by a single mild injury in both brain and peripheral blood leukocytes in a severity- and time-dependent manner. Moreover, we were able to demonstrate that specific inhibitors of the CtBPs effectively suppress the expression of the CtBP target genes and thus improve neurological outcome in mice receiving single and repeated mild TBIs. This discovery suggests new avenues for therapeutic modulation of the inflammatory response to brain injury.

neuroscience↗

bioRxiv2019

Rapidly cycling stem cells regenerate the intestine independent of Lgr5high cells

The +4 cells in intestinal crypts are DNA damage-resistant and contribute to regeneration. However, their exact identity and the mechanism underlying +4 cell-mediated regeneration remain unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are enriched at the +4 position in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radio-sensitive crypt base columnar stem cells (CBCs); they enable fast repopulation of the intestinal epithelium independent of CBCs that are largely depleted after irradiation. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are rapidly cycling ISCs that maintain and regenerate the intestinal epithelium.

cell biology↗

bioRxiv2019

Genomic expansion of archaeal lineages resolved from deep Costa Rica sediments

Numerous archaeal lineages are known to inhabit marine subsurface sediments, although their distributions, metabolic capacities and interspecies interactions are still not well understood. Abundant and diverse archaea were recently reported in Costa Rica (CR) margin subseafloor sediments recovered during IODP Expedition 334. Here, we recover metagenome-assembled genomes (MAGs) of archaea from the CR-margin and compare them to their relatives from shallower settings. We describe 31 MAGs of 6 different archaeal lineages (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales and Hadesarchaea) and thoroughly analyze representative MAGs from the phyla Lokiarchaeota and Bathyarchaeota. Our analysis suggests the potential capabilities of Lokiarchaeota members to anaerobically degrade aliphatic and aromatic hydrocarbons. We show it is genetically possible and energetically feasible for Lokiarchaeota to degrade benzoate if they associate with organisms using nitrate, nitrite and sulfite as electron acceptors, which suggests a possibility of syntrophic relationships between Lokiarchaeota and nitrite and sulfite reducers. The novel Bathyarchaeota lineage possesses an incomplete methanogenesis pathway lacking the methyl co-enzyme M reductase complex and encodes a non-canonical acetogenic pathway potentially coupling methylotrophy to acetogenesis via the methyl branch of Wood-Ljundahl pathway. These novel metabolic characteristics suggest the potential of this Bathyarchaeota lineage to be a transition between methanogenic and acetogenic Bathyarchaeota lineages. This work substantially expands our knowledge about the metabolic function repertoire of marine benthic archaea.

microbiology↗

bioRxiv2019

In situ growth of anammox bacteria in subseafloor sediments

The deep biosphere buried in marine sediments was estimated to host an equal number of microbes as found in the above oceans 1. It has been debated if these cells are alive and active 2, and their per cell energy availability does not seem to allow for net population growth 3. Here, we report the growth of anammox bacteria in [~]80,000 year old subsurface sediments indicated by their four orders of magnitude abundance increase in the nitrate-ammonia transition zone (NATZ). Their growth coincides with a local increase in anammox power supply. The genome of the dominant anammox bacterium from the NATZ was reconstructed and showed an increased index of replication confirming in situ active growth. The genome belongs to a new Scalindua species so far exclusively found in marine environments, which has the genetic capacity of urea and cyanate utilization and is enriched in genes allowing it to cope with external environmental stressors, such as energy limitation. Our results suggest that specific microbial groups are not only able to survive over geological timescales, but also thrive in the deep subsurface when encountering favorable conditions.

microbiology↗

bioRxiv2019

Transgenerational inheritance of betaine-induced epigenetic alterations in estrogen-responsive IGF-2/IGFBP2 genes in rat hippocampus

Betaine as a methyl donor participates in DNA methylation. Here, we sought to investigate the effects of betaine on hippocampal expression of neurogenesis genes and their DNA methylation status across three generations. Pregnant rats (F0) were fed control and betaine-supplemented diets throughout gestation and lactation; and female F1 and F2 offspring at weaning, together with the F0 dams, were used in the study. Hippocampal expression of aromatase, estrogen receptor and estrogen related receptor {beta} was down-regulated in F1, together with the estrogen-responsive IGF-2/IGFBP2 genes. However, all these genes were up-regulated in F2, which follows the same pattern of F0. In agreement with changes in mRNA expression, the imprinting control region (ICR) of IGF-2 gene was hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the promoter DNA methylation status of all the affected genes was hypermethylated in F1 but hypomethylated in F2 and F0. Betaine homocysteine methyltransferase followed same pattern of transgenerational inheritance. These results indicate that betaine exerts a transgenerational effect on hippocampal expression of estrogen-responsive genes in rat offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes.\n\nAuthor SummaryEpigenetic transgenerational inheritance is defined as the phenotypic change across generations and germline transmission of epigenetic information without direct genetic manipulation or environmental exposure. Maternal and paternal diet has been shown to have transgenerational behavioral and physiological effects. Maternal intake of methyl donors, including folate, betaine, and choline during different gestation period alters epigenetic modifications of many imprinted genes in the genomic DNA isolated from cord blood. Betaine as a methyl donor nutrition plays an important role in brain development. However, the effect of maternal betaine supplementation on transgenerational inheritance of imprinted gene IGF-2 in offspring hippocampus is still unknown. Here, we sought to investigate the effects of betaine on rat hippocampal expression of neurogenesis genes and their DNA methylation status across three generations. We found that betaine significantly decreased estrogen-responsive genes IGF-2/IGFBP2 expression in rat F1 offspring, yet increased in F2 offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes. Our results suggesting that maternal methyl donor nutrition supplementation may be exert an epigenetic transgenerational effect on female offspring.

genetics↗

bioRxiv2019

OxyR senses reactive sulfane sulfur and activates genes for its removal in Escherichia coli

Reactive sulfane sulfur species such as hydrogen polysulfide and organic persulfide are newly recognized as normal cellular components, involved in signaling and protecting cells from oxidative stress. Their production is extensively studied, but their removal is less characterized. Herein, we showed that reactive sulfane sulfur is toxic at high levels, and it is mainly removed via reduction by thioredoxin and glutaredoxin with the release of H2S in Escherichia coli. OxyR is best known to respond to H2O2, and it also played an important role in responding to reactive sulfane sulfur under both aerobic and anaerobic conditions. It was modified by hydrogen polysulfide to OxyR C199-SSH, which activated the expression of thioredoxin 2 and glutaredoxin 1. This is a new type of OxyR modification. Bioinformatics analysis showed that OxyRs are widely present in bacteria, including strict anaerobic bacteria. Thus, the OxyR sensing of reactive sulfane sulfur may represent a conserved mechanism for bacteria to deal with sulfane sulfur stress.

microbiology↗

bioRxiv2019

MetFish: A Metabolomics Platform for Studying Microbial Communities in Chemically Extreme Environments

Metabolites have essential roles in microbial communities, including as mediators of nutrient and energy exchange, cell-to-cell communication, and antibiosis. However, detecting and quantifying metabolites and other chemicals in samples having extremes in salt or mineral content using liquid chromatography-mass spectrometry (LC-MS)-based methods remains a significant challenge. Here we report a facile method based on in situ chemical derivatization followed by extraction for analysis of metabolites and other chemicals in hypersaline samples, enabling for the first time direct LC-MS-based exo-metabolomics analysis in sample matrices containing up to 2 molar total dissolved salts. The method, MetFish, is applicable to molecules containing amine, carboxylic acid, carbonyl, or hydroxyl functional groups, and can be integrated into either targeted or untargeted analysis pipelines. In targeted analyses, MetFish provided limits of quantification as low as 1 nM, broad linear dynamic ranges (up to 5-6 orders of magnitude) with excellent linearity, and low median inter-day reproducibility (e.g. 2.6%). MetFish was successfully applied in targeted and untargeted exo-metabolomics analyses of microbial consortia, quantifying amino acid dynamics in the exo-metabolome during community succession; in situ in a native prairie soil, whose exo-metabolome was isolated using a hypersaline extraction; and in input and produced fluids from a hydraulically fractured well, identifying dramatic changes in the exo-metabolome over time in the well.

microbiology↗

bioRxiv2018

Functional importance of JMY expression by Sertoli cells in mediating mouse spermatogenesis

Sertoli cells are crucial for spermatogenesis in the seminiferous epithelium because their actin cytoskeleton supports vesicle transport, cell junction, protein anchoring and spermiation. Here, we show that junction-mediating and regulatory protein (JMY), an actin regulating protein, also affects endocytic vesicle trafficking and Sertoli cell junction remodeling since disruption of these functions induced male subfertility in Sertoli cell-specific Jmy knockout mice. Specifically, these mice have: a) impaired BTB integrity and spermatid adhesion in the seminiferous tubules; b) high incidence of sperm structural deformity; c) reduced sperm count and poor sperm motility. Moreover, the cytoskeletal integrity in Sertoli cell-specific Jmy knockout mice was compromised along with endocytic vesicular trafficking. These effects impaired junctional protein recycling and reduced Sertoli cell junctions. In addition, JMY interaction with -actinin1 and Sorbs2 was related to JMY activity and in turn actin cytoskeletal organization. In summary, JMY affects control of spermatogenesis through regulating actin filament organization and endocytic vesicle trafficking in Sertoli cells.

cell biology↗

bioRxiv2018

De novo computational RNA modeling into cryoEM maps of large ribonucleoprotein complexes

RNA-protein assemblies carry out many critical biological functions including translation, RNA splicing, and telomere extension. Increasingly, cryo-electron microscopy (cryoEM) is used to determine the structures of these complexes, but nearly all maps determined with this method have regions in which the local resolution does not permit manual coordinate tracing. Because RNA coordinates typically cannot be determined by docking crystal structures of separate components and existing structure prediction algorithms cannot yet model RNA-protein complexes, RNA coordinates are frequently omitted from final models despite their biological importance. To address these omissions, we have developed a new framework for De novo Ribonucleoprotein modeling in Real-space through Assembly of Fragments Together with Electron density in Rosetta (DRRAFTER). We show that DRRAFTER recovers near-native models for a diverse benchmark set of small RNA-protein complexes, as well as for large RNA-protein machines, including the spliceosome, mitochondrial ribosome, and CRISPR-Cas9-sgRNA complexes where the availability of both high and low resolution maps enable rigorous tests. Blind tests on yeast U1 snRNP and spliceosomal P complex maps demonstrate that the method can successfully build RNA coordinates in real-world modeling scenarios. Additionally, to aid in final model interpretation, we present a method for reliable in situ estimation of DRRAFTER model accuracy. Finally, we apply this method to recently determined maps of telomerase, the HIV-1 reverse transcriptase initiation complex, and the packaged MS2 genome, demonstrating that DRRAFTER can be used to accelerate accurate model building in challenging cases.

biophysics↗

bioRxiv2018

Next generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic over-expression genetic analysis in Drosophila melanogaster. We present flySAM, a potent new tool for in vivo CRISPRa, which offers a major improvement over existing strategies in terms of effectiveness, scalability, and ease-of-use. flySAM outperforms existing in vivo CRISPRa strategies, and approximates phenotypes obtained using traditional Gal4-UAS over-expression. Further, because flySAM typically only requires a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental usage of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will thus replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic over-expression resource, TRiP-OE.

genetics↗

bioRxiv2017

Map-based cloning of the gene Pm21 that confers broad spectrum resistance to wheat powdery mildew

Common wheat (Triticum aestivum L.) is one of the most important cereal crops. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a continuing threat to wheat production. The Pm21 gene, originating from Dasypyrum villosum, confers high resistance to all known Bgt races and has been widely applied in wheat breeding in China. In this research, we identify Pm21 as a typical coiled-coil, nucleotide-binding site, leucine-rich repeat gene by an integrated strategy of resistance gene analog (RGA)-based cloning via comparative genomics, physical and genetic mapping, BSMV-induced gene silencing (BSMV-VIGS), large-scale mutagenesis and genetic transformation.

genetics↗

bioRxiv2017

Extensive hidden genetic variation shapes the structure of functional elements in Drosophila

Mutations that add, subtract, rearrange, or otherwise refashion genome structure often affect phenotypes, though the fragmented nature of most contemporary assemblies obscure them. To discover such mutations, we assembled the first reference quality genome of Drosophila melanogaster since its initial sequencing. By comparing this genome to the existing D. melanogaster assembly, we create a structural variant map of unprecedented resolution, revealing extensive genetic variation that has remained hidden until now. Many of these variants constitute strong candidates underlying phenotypic variation, including tandem duplications and a transposable element insertion that dramatically amplifies the expression of detoxification genes associated with nicotine resistance. The abundance of important genetic variation that still evades discovery highlights how crucial high quality references are to deciphering phenotypes.

genomics↗