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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 595 records · Page 33Linked to original sources

bioRxiv2017

Environmental DNA metabarcoding of rivers: Not all eDNA is everywhere, and not all the time

Environmental DNA metabarcoding has become a popular tool for the assessment of freshwater biodiversity, but it is largely unclear how sampling time and location influence the assessment of communities. Abiotic factors in rivers can change on small spatial and temporal scale and might greatly influence eDNA metabarcoding results. In this study, we sampled three German rivers at four locations per sampling site: 1. Left river side, surface water 2. Right river side, surface water, 3. Left side, close to the riverbed, 4. Right side, close to the riverbed. For the rivers Ruhr and Mohne, sampling was conducted three times in spring, each sampling one week apart. The Ruhr was again sampled in autumn and the Gillbach was sampled in winter. Sequencing on an Illumina MiSeq with COI primers Bf2/BR2 revealed diverse communities (6493 Operational taxonomic units, OTUs), which largely differed between rivers. Communities changed significantly over time in the Ruhr, but not in the Mohne. Sampling location influenced recovered communities in the Mohne and in the Ruhr in autumn. Our results have important implications for future eDNA studies, which should take into account that not all eDNA in rivers is everywhere and not at all times.

molecular biology↗

bioRxiv2017

siRNA biogenesis, DNA methylation and target locus silencing are distinct and separable Pol IV sub-functions differentially dependent on the largest subunit's CTD

Plant nuclear multisubunit RNA polymerase IV plays a key role in the RNA-directed DNA methylation (RdDM) pathway for transcriptional silencing of transposons, viruses and specific genes by synthesizing precursors of 24 nt siRNAs that guide the process. The Pol IV largest subunit, NRPD1 is derived from the Pol II largest subunit but has a unique carboxy-terminal domain (CTD) of unknown function. We show that the NRPD1 CTD is critical for transcriptional silencing of target loci and for producing 24 nt siRNAs at high levels. However, the CTD is surprisingly dispensable for near wild-type levels of Pol IV-dependent genomic cytosine methylation. These results suggest that low levels of 24 nt siRNAs, produced at only 20-30% of wild-type levels, are sufficient for full RNA-directed DNA methylation, yet insufficient for silencing, suggesting additional roles for siRNAs beyond DNA methylation. Moreover, at a subset of target loci, neither siRNA levels nor cytosine methylation are impaired upon deletion of the CTD, yet silencing is lost. Collectively, the non-linear relationships between siRNA levels, cytosine methylation and silencing suggest the existence of additional mechanisms of silencing dependent on Pol IV transcription and mediated by the CTD, such as promoter occlusion to inhibit the activities of other polymerases.

molecular biology↗

bioRxiv2017

The Cryo-Electron Microscopy Structure of the Type 1 Chaperone-Usher Pilus Rod

Chaperone-usher pili are long, polymeric protein fibres displayed on the surface of many bacterial pathogens. These critical virulence factors allow bacteria to specifically attach to host cells during infection. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during UPECs colonisation of the urinary tract, mediating bacterial attachment to the bladder and kidney, respectively. Also, their biomechanical properties that allow them to reversibly uncoil in response to flow-induced forces are critical for UPECs ability to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2 [A] resolution cryo-electron microscopy (cryo-EM) structure of the type 1 pilus rod, which together with the previous structure of the P pilus rod, enables us to understand the remarkable \"spring-like\" properties of chaperone-usher pili in more detail.

molecular biology↗

bioRxiv2017

Double-strand breaks are not the main cause of spontaneous sister chromatid exchange in wild-type yeast cells

SummaryHomologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. Contrary to what is commonly thought, we find that most spontaneous SCE events are not due to the repair of DNA double-strand breaks.

molecular biology↗

bioRxiv2017

Full-length mRNA sequencing uncovers a widespread coupling between transcription and mRNA processing

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we found 2,700 genes with interdependent alternative transcription, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules. The analysis of three human primary tissues revealed similar patterns of interdependency between transcription and mRNA processing events. We predict thousands of novel Open Reading Frames from the sequence of full-length mRNAs and obtained evidence for their translation by shotgun proteomics. The mapping database rescued 358 previously unassigned peptides and improved the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improved proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription and mRNA processing.

molecular biology↗

bioRxiv2017

Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts separated by a boundary or hinge region. Using in situ DNase Hi-C in mouse cells with deletions or inversions within the hinge we show that the conserved repeat locus Dxz4 alone is sufficient to maintain the bipartite structure and that Dxz4 orientation controls the distribution of long-range contacts on the Xi. Frequent long-range contacts between Dxz4 and the telomeric superdomain are either lost after its deletion or shifted to the centromeric superdomain after its inversion. This massive reversal in contact distribution is consistent with the reversal of CTCF motif orientation at Dxz4. De-condensation of the Xi after Dxz4 deletion is associated with partial restoration of TADs normally attenuated on the Xi. There is also an increase in chromatin accessibility and CTCF binding on the Xi after Dxz4 deletion or inversion, but few changes in gene expression, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of a bank of CTCF motifs at Dxz4, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

molecular biology↗

bioRxiv2017

Transcription start site profiling uncovers divergent transcription and enhancer-associated RNAs in Drosophila melanogaster

BackgroundHigh-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis.\n\nResultsThe data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for \"enhancer RNAs\" (eRNAs) in defining transcriptional programs that are important for animal development.\n\nConclusionsHigh-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.

molecular biology↗

bioRxiv2017

SOD1A4V aggregation alters ubiquitin homeostasis in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving the selective death of upper and lower motor neurons in the primary motor cortex and spinal cord. A hallmark of ALS pathology is the accumulation of ubiquitinated protein inclusions within motor neurons. Previous studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here we show that Ub homeostasis is altered in a SOD1 cell model of ALS. Utilising fluorescently tagged Ub, we followed the distribution of Ub in living cells expressing SOD1 and show that Ub is present at the earliest stages of SOD1 aggregation. We also report that cells containing aggregates of mutant SOD1 have greater ubiquitin-proteasome system (UPS) dysfunction as measured by the accumulation of the fluorescent proteasome reporter tdTomatoCL1. Furthermore, SOD1 aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that mutant SOD1 is associated with a shift of Ub to a pool of supersaturated proteins including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.

molecular biology↗

bioRxiv2017

Expanding the Atlas of Functional Missense Variation for Human Genes

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon-mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features, and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

molecular biology↗

bioRxiv2017

The transcriptionally permissive chromatin state of ES cells is acutely tuned totranslational output

A permissive chromatin environment coupled to hypertranscription is critical to drive the rapid proliferation of Embryonic Stem (ES) cells and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ES cells. The results reveal that activity of cellular growth pathways, prominently protein synthesis, perpetuates the euchromatic state and hypertranscription of ES cells. Acute, mild inhibition of translation results in rapid depletion of euchromatic marks in ES cells and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Remarkably, reduced translational output leads to rewiring of open chromatin within 3 hours, including decreased accessibility at a subset of active developmental enhancers and increased accessibility at histone genes and transposable elements. Using a proteome-scale analysis, we show that several euchromatin regulators are unstable proteins and thus continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription and translation sets the pace of proliferation at peri-implantation and may be employed generally by stem/progenitor cells.

molecular biology↗

bioRxiv2017

Modest static pressure suppresses columnar epithelial cell proliferation in association with cell shape and cytoskeletal modifications

Intraluminal pressure elevation can cause degenerative disorders, such as ileus and hydronephrosis, and the threshold is fairly low and constant, 20-30 cm H2O. We previously devised a novel two-chamber culture system subjecting cells cultured on a semipermeable membrane to increased culture medium height (water pressure up to 60 cm H2O). Here, we cultured several different cell lines using the low static pressure-loadable two-chamber system, and examined cell growth, cell cycle, and cell morphology. Madin-Darby canine kidney (MDCK) columnar epithelial cells were growth-suppressed in a manner dependent on static water pressure ranging from 2-50 cm H2O, without cell cycle arrest at any specific phase. Two other types of columnar epithelial cells exhibited similar phenotypes. By contrast, spherical epithelial and mesenchymal cells were not growth-suppressed, even at 50 cm H2O. Phalloidin staining revealed that 50 cm H2O pressure load vertically flattened and laterally widened columnar epithelial cells and made actin fiber distribution sparse, without affecting total phalloidin intensity per cell. When the mucosal protectant irsogladine maleate (100 nM) was added to 50-cm-high culture medium, MDCK cells were reduced in volume and their doubling time shortened. Cell proliferation and morphology are known to be regulated by the Hippo signaling pathway, but a pressure load of 50 cm H2O did not alter the expression levels of Hippo signaling molecules in columnar epithelial cells, suggesting that this pathway was not involved in the pressure-induced phenotypes. RNA sequencing of MDCK cells showed that a 50 cm H2O pressure load upregulated keratin 14, an intermediate filament, 12-fold. This upregulation was confirmed at the protein level by immunofluorescence, suggesting a role in cytoskeletal reinforcement. These results provide evidence that cell morphology and the cytoskeleton are closely linked to cell growth. Pathological intraluminal pressure elevation may cause mucosal degeneration by acting directly on this linkage.\n\nSummaryWe provide evidence that columnar epithelial cells are growth-suppressed by pressure loads as low as 30 cm H2O, in association with cell-shape flattening and cytoskeletal alterations.

molecular biology↗

bioRxiv2017

Evaluation of Topramezone on Zebrafish Retinoid Signaling

Topramezone is a highly selective herbicide developed for broadleaf and grass weeds control in corn. In this study, the effects of topramezone on zebrafish, especially in retinoid signaling were investigated. Zebrafish embryos were treated with topramezone from 4 hours post-fertilization (hpf) to 144 hpf. Exposed to topramezone significantly reduced the retinal and retinoic levels compared to controls. The transcriptional expression levels of retinol dehydrogenase (rdh1), retinoic acid receptor subunit (raraa), retinal dehydrogenase (raldh2), retinol binding protein (rbp1a), and cellular retinoic acid binding protein (crabp1a and crabp2a) were significantly decreased. Our results suggested that topramezone significantly impaired zebrafish retinoid signaling during a short time exposure. However, treatment with topramezone significantly increased the mRNA expression levels of zfblue, zfrho, zfgr1, zfuv, and zfred. Our data demonstrated that topramezone treatment could interrupt retinoid signaling and further affect zebrafish eye development.

molecular biology↗

bioRxiv2017

Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

Telomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)-) TLC1 isoform. Here, we first show that Sm7 can perform this function even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we tested the hypothesis that the location of the Sm7-binding site relative to the 3' end is contrastingly important. When we moved the Sm site to locations 5' of its native position, we observed that this stabilized shorter forms of poly(A)- TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm7 binding to TLC1 controls where the mature poly(A)- 3' end is formed. In summary, our results show that Sm7 and the 3' end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

molecular biology↗

bioRxiv2017

The Correlation between MicroRNA-199a and White Adipose Tissue in C57/BL6J Mice with High-Fat Diet

Understanding is emerging about microRNAs as mediators in the regulation of white adipose tissue (WAT) and obesity. The expression level of miR-199a in mice was investigated to test our hypothesis: miR-199a might be related to fat accumulation and try to find its target mRNA, which perhaps could propose strategies with a therapeutic potential affecting the fat storage. C57/BL6J mice were randomly assigned to either a control group or an obesity model group (n=10 in both groups). Control mice were fed a normal diet (fat: 10 kcal %) ad libitum for 12 weeks, and model mice were fed a high-fat diet (fat: 30 kcal %) ad libitum for 12 weeks to induce obesity. At the end of the experiment, body fat mass and the free fatty acids (FFAs) and triglycerides (TGs) in WAT were tested. Fat cell size was measured by hematoxylin-eosin (H&E) staining method. The fat mass of the model group was higher than that of the control group (P<0.05). In addition, the concentrations of the FFAs and TGs were higher (P<0.05) and the adipocyte count was lower (P<0.05) in the model group. We tested the expression levels of miR-199a and key adipogenic transcription factors, including peroxisome proliferator activated receptor gamma2 (PPAR{gamma}2), CCAAT/enhancer binding proteins alpha (C/EBP), adipocyte fatty acid-binding protein (aP2), and sterol regulatory element binding protein-1c (SREBP-1c). Up-regulated expression of miR-199a was observed in model group. Increased levels of miR-199a was accompanied by high expression levels of SREBP-1c. We found that the 3-UTR of SREBP-1c mRNA has a predicted binding site for miR-199a. Based on the current discoveries, we suggest that miR-199a may exert its action by binding to its target mRNA and cooperate with SREBP-1c to induce obesity. Therefore, if the predicted binding site is confirmed by further research, miR-199a may have therapeutic potential for obesity.\n\nAbbreviationsWAT, white adipose tissue; PPAR{gamma}2, peroxisome proliferator, activated receptor {gamma}2; C/EBP CCAAT/enhancer binding proteins ; aP2, adipocyte fatty acid-binding protein; SREBP-1c, sterol regulatory element binding protein-1c; HFD, high-fat diet.

molecular biology↗

bioRxiv2017

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

CRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response is tp53-dependent and the toxic effect of tp53 severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor for tp53 function, especially in hPSCs which spontaneously acquire tp53 mutations.

molecular biology↗

bioRxiv2017

High throughput amplicon sequencing to assess within- and between-host genetic diversity in plant viruses

Molecular epidemiology approaches at the landscape scale require to study the genetic diversity of viral populations from numerous hosts and to characterize mixed infections. In such a context, high-throughput amplicon sequencing (HTAS) techniques create interesting opportunities as they allow identifying distinct variants within a same host while simultaneously genotyping a high number of samples. Validating variants produced by HTAS may, however, remain difficult due to biases occurring at different steps of the data-generating process (e.g. environmental contaminations and sequencing error). Here, we focused on Endive necrotic mosaic virus (ENMV), a member of family Potyviridae, genus Potyvirus to develop an HTAS approach and to characterize the genetic diversity at the intra- and inter-host levels from 430 samples collected over an area of 1660 km2 located in south-eastern France. We demonstrated how it is possible, by incorporating various controls in the experimental design and by performing independent sample replicates, to estimate potential biases in HTAS results and to implement an automated and robust variant calling procedure.\n\nHighlightsO_LIHigh-throughput amplicon sequencing to assess plant virus genetic diversity\nC_LIO_LIEstimating bias in high throughput amplicon sequencing results\nC_LIO_LIAutomated variant calling procedure for robust high throughput amplicon sequencing\nC_LI

molecular biology↗

bioRxiv2017

INO80 Chromatin Remodelling Coordinates Metabolic Homeostasis with Cell Division

Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the Yeast Metabolic Cycle (YMC). Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here we demonstrate that the INO80 chromatin-remodelling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin in order to restrict proliferation to metabolically optimal states.

molecular biology↗

bioRxiv2017

Identification of disease resistance genes from a Chinese wild grapevine (Vitis davidii) by analysing grape transcriptomes and transgenic Arabidopsis

HighlightTranscription profiles showed that 20 candidate genes were obviously co-expressed at 12 hpi in 30 Vitis davidii.\n\nVdWRKY53 trancription factor enhanced the resisitance in grapevine and Arabidopsis.\n\nAbstractThe molecular mechanisms underlying disease tolerance in grapevines remain uncharacterized, even though there are substantial differences in the resistance of grapevine species to fungal and bacterial diseases. In this study, we identified genes and genetic networks involved in disease resistance in grapevines by comparing the transcriptomes of a strongly resistant clone of Chinese wild grapevine (Vitis davidii cv. Ciputao 941, DAC) and a susceptible clone of European grapevine (Vitis vinifera cv. Manicure Finger, VIM) before and after infection with white rot disease (Coniella diplodiella). Disease resistance-related genes were triggered in DAC approximately 12 hours post infection (hpi) with C. diplodiella. Twenty candidate resistant genes were co-expressed in DAC. One of these candidate genes, VdWRKY53 (GenBank accession KY124243), was over-expressed in transgenic Arabidopsis thaliana plants and was found to provide these plants with enhanced resistance to C. diplodiella, Pseudomonas syringae pv tomato PDC3000, and Golovinomyces cichoracearum. This result indicates that VdWRKY53 may be involved in nonspecific resistance via interaction with fungal and oomycete elicitor signals and the activation of defence gene expression. These results provide potential gene targets for molecular breeding to develop resistant grape cultivars.

molecular biology↗