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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 451 records · Page 25Linked to original sources

bioRxiv2020

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has driven fundamental advances in cell and molecular biology, enabling researchers to introduce precise mutations, generate fluorescent protein fusions for localization and to confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size and particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, with a high percentage of genes containing complex sequence structures, an average genomic GC content of 64% and gene expression requiring regular introns for stable transcription. Here we overcome these challenges via the development of a recombineering pipeline that enables the rapid parallel cloning of genes from a Chlamydomonas BAC collection. We show the method can successfully retrieve large and complex genes that PCR-based methods have previously failed to clone, including genes as large as 23 kilobases, thus making previously technically challenging genes to study now amenable to cloning. We initially applied the pipeline to 12 targets with a 92% cloning success rate. We then developed a high-throughput approach and targeted 191 genes relating to the Chlamydomonas CO2 concentrating mechanism (CCM) with an overall cloning success rate of 77% that is independent of gene size. Localization of a subset of CCM targets has confirmed previous mass spectrometry data and identified new pyrenoid components. To expand the functionality of our system, we developed a series of localization vectors that enable complementation of Chlamydomonas Library Project mutants and enable protein tagging with a range of fluorophores. Vectors and detailed protocols are available to facilitate the easy adoption of this method by the Chlamydomonas research community. We envision that this technology will open up new possibilities in algal and plant research and be complementary to the Chlamydomonas mutant library.

plant biology↗

bioRxiv2020

Computational analysis of ligand dose range thermal proteome profiles

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. We present a statistical analysis method with reliable control of the false discovery rate and apply it to several datasets probing epigenetic drugs. We detect off-target drug engagement in unrelated protein families.

bioinformatics↗

bioRxiv2020

A putative new SARS-CoV protein, 3a*, encoded in an ORF overlapping ORF3a

Identification of the full complement of genes in SARS-CoV-2 is a crucial step towards gaining a fuller understanding of its molecular biology. However, short and/or overlapping genes can be difficult to detect using conventional computational approaches, whereas high throughput experimental approaches - such as ribosome profiling - cannot distinguish translation of functional peptides from regulatory translation or translational noise. By studying regions showing enhanced conservation at synonymous sites in alignments of SARS-CoV and related viruses (subgenus Sarbecovirus), and correlating with the conserved presence of an open reading frame and plausible translation mechanism, we identified a putative new gene, ORF3a*, overlapping ORF3a in an alternative reading frame. A recently published ribosome profiling study confirmed that ORF3a* is indeed translated during infection. ORF3a* is conserved across the subgenus Sarbecovirus, and encodes a 40-41 amino acid predicted transmembrane protein.

bioinformatics↗

bioRxiv2020

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for AMPA-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a new method for the rapid and selective labeling of chemical probes to AMPARs by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological conditions. This method allowed us to quantify AMPAR distribution and trafficking, which revealed some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method was also successfully expanded to selectively label NMDA-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

biochemistry↗

bioRxiv2020

Introducing undergraduate students to reverse genetics in the laboratory: a case study of a project-oriented learning (POL) system in Taishan Medical University, China

Biochemistry and molecular biology have become increasingly reliant on experimental experience, because they are difficult subjects to understand from a theoretical approach. The Innovation and Entrepreneurship Training Program was established by the China Ministry of Education to foster the scientific interests and experiences of undergraduate students. Here, we reported on an innovative project-oriented learning (POL) system directed by Taishan Medical University (Named Shandong First Medical University & Shandong Academy of Sciences since February 2019) China, which fused innovative project with biochemical course concepts. This study takes reverse genetics, which is considered to be difficult to understand through course-only learning, as an example. To help undergraduate students understand the principle of reverse genetics, the project involved producing hepatitis C virus in vitro. Furthermore, to give them a comprehensive scientific experience based on their knowledge from textbook, extensive mentorship was provided through the project, including theoretical guidance and technical support for experiments. Assessment showed a comprehensive improvement after participating in these programs. In conclusion, this POL system was a good model that enabled undergraduates to learn more during courses. It also provided a chance to experience hands-on sciences, which might motivate them to pursue a career in science.View Full Text

scientific communication and education↗

bioRxiv2020

Protein structure search to support the development of protein structure prediction methods

Protein structure prediction is a long-standing unsolved problem in molecular biology that has seen renewed interest with the recent success of deep learning with AlphaFold at CASP13. While developing and evaluating protein structure prediction methods, researchers may want to identify the most similar known structures to their predicted structures. These predicted structures often have low sequence and structure similarity to known structures. We show how RUPEE, a purely geometric protein structure search, is able to identify the structures most similar to structure predictions, regardless of how they vary from known structures, something existing protein structure searches struggle with. RUPEE accomplishes this through the use of a novel linear encoding of protein structures as a sequence of residue descriptors. Using a fast Needleman-Wunsch algorithm, RUPEE is able to perform alignments on the sequences of residue descriptors for every available structure. This is followed by a series of increasingly accurate structure alignments from TM-align alignments initialized with the Needleman-Wunsch residue descriptor alignments to standard TM-align alignments of the final results. By using alignment normalization effectively at each stage, RUPEE also can execute containment searches in addition to full-length searches to identify structural motifs within proteins. We compare the results of RUPEE to mTM-align, SSM, CATHEDRAL and VAST using a benchmark derived from the protein structure predictions submitted to CASP13. RUPEE identifies better alignments on average with respect to RMSD and TM-score as well as Q-score and SSAP-score, scores specific to SSM and CATHEDRAL, respectively. Finally, we show a sample of the top-scoring alignments that RUPEE identified that none of the other protein structure searches we compared to were able to identify. The RUPEE protein structure search is available at https://ayoubresearch.com. Code and data are available at https://github.com/rayoub/rupee.

bioinformatics↗

bioRxiv2020

Enhanced production of taxadiene in Saccharomyces cerevisiae

Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at shake flask scale. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively. Maximum taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results highlight the positive effect of coupling molecular biology tools with bioprocess variable optimisation on synthetic pathway development.HighlightsMaximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °CIntegrating fusion protein tagged-taxadiene synthase improved taxadiene titre.Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Apertor Labs, Ansa Biotechnologies, and Berkeley Brewing Sciences.AbbreviationsBTS1Geranylgeranyl diphosphate synthasecrtEGeranylgeranyl diphosphate synthaseDMAPPDimethylallyl pyrophosphateERG8Phosphomevalonate kinaseERG9Farnesyl-diphosphate farnesyl transferase (squalene synthase)ERG103-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthaseERG12Mevalonate kinaseERG133-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthaseERG19Mevalonate pyrophosphate decarboxylaseERG20Farnesyl pyrophosphate synthetaseFPPFarnesyl diphosphateGGOH(E,E,E)-geranylgeraniolGGPP(E,E,E)-Geranylgeranyl diphosphateGPPGeranyl diphosphateHMG13-hydroxy-3-methylglutaryl-coenzyme A reductase 1HMG23-hydroxy-3-methylglutaryl-coenzyme A reductase 2IDIIsopentenyl-diphosphate delta-isomeraseIPPIsopentenyl pyrophosphateMBPMaltose binding proteinmvaEAcetyl-CoA acetyltransferasemvaSHydroxymethylglutaryl-CoA synthaseMVA pathwayMevalonate pathwayTASYTaxadiene synthase.View Full Text

synthetic biology↗

bioRxiv2020

Optimized culture of retinal ganglion cells and amacrine cells from adult mice

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSC). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500{micro}m in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model adult neurons in vivo. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.

neuroscience↗

bioRxiv2020

Puromycin reactivity does not accurately localize translation at the subcellular level

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Here, using in vitro and in vivo experiments, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in the in vivo context.

biochemistry↗

bioRxiv2020

openPFGE: An open source and low cost pulsed-field gel electrophoresis equipment

DNA electrophoresis is a fundamental technique in molecular biology that allows the separation of DNA molecules up to ~50 Kbp. Pulsed-field gel electrophoresis [PFGE] is a variation of the conventional DNA electrophoresis technique that allows the separation of very large DNA molecules up to ~10 Mbp. PFGE equipment is very expensive and it becomes an access barrier to many laboratories. Also, just a few privative designs of the equipment are available and it becomes difficult for the community to improve or customize their functioning. Here, we provide an open source PFGE equipment capable of the separation of DNA molecules up to, at least, ~2 Mbp and at low cost: USD$850, about 3% of the price of typical commercial equipment. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@3e8d8eorg.highwire.dtl.DTLVardef@bf96fforg.highwire.dtl.DTLVardef@525546org.highwire.dtl.DTLVardef@15c6aeeorg.highwire.dtl.DTLVardef@705b0a_HPS_FORMAT_FIGEXP M_TBL C_TBL

biochemistry↗

bioRxiv2020

A low-cost platform suitable for sequencing-based recovery of natural variation in understudied plants.

Genetic characterization of wild and cultivated plants provides valuable knowledge for conservation and agriculture. DNA sequencing technologies are improving and costs are dropping. Yet, analysis of many species is hindered because they grow in regions that lack infrastructure for advanced molecular biology. We developed and adapted low-cost methods that address these issues. Tissue is collected and stored in silica-gel, avoiding the need for liquid nitrogen and freezers. We have optimized low-cost home-made DNA extraction to increase yields, reduce costs, and produce DNA suitable for next generation sequencing. We also describe how to build a gel documentation system for DNA quantification. As a proof of principle, we use these methods to evaluate wild Berberis darwinii, native to Southern Chile.Method summary We describe a suite of low-cost do-it-yourself methods for field collection of plant tissues, extraction of genomic DNA suitable for next generation sequencing, and home-made agarose gel documentation suitable for DNA quantification. These methods enable the collection and preparation of samples for genomic analysis in regions with limited infrastructure.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Nested Stochastic Block Models Applied to the Analysis of Single Cell Data

Single cell profiling has been proven to be a powerful tool in molecular biology to understand the complex behaviours of heterogeneous system. The definition of the properties of single cells is the primary endpoint of such analysis, cells are typically clustered to underpin the common determinants that can be used to describe functional properties of the cell mixture under investigation. Several approaches have been proposed to identify cell clusters; while this is matter of active research, one popular approach is based on community detection in neighbourhood graphs by optimisation of modularity. In this paper we propose an alternative and principled solution to this problem, based on Stochastic Block Models. We show that such approach not only is suitable for identification of cell groups, it also provides a solid framework to perform other relevant tasks in single cell analysis, such as label transfer. To encourage the use of Stochastic Block Models, we developed a python library, schist, that is compatible with the popular scanpy framework.

bioinformatics↗

bioRxiv2020

NGS-PrimerPlex: high-throughput primer design for multiplex polymerase chain reactions

SummaryMultiplex PCR has multiple applications in molecular biology, including developing new targeted NGS-panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier and extension of existing NGS-panels. The primer design process takes into consideration formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome SNPs overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with median coverage of 97.4% of target regions by at least 30 reads. Availability and implementationNGS-PrimerPlex is open-source and freely available at https://github.com/aakechin/NGS-PrimerPlex/. Contactaa_kechin@niboch.nsc.ru Supplementary informationSupplementary data

bioinformatics↗

bioRxiv2020

Light into the darkness: Unifying the known and unknown coding sequence space in microbiome analyses

Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40%-60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we propose a conceptual framework and a computational workflow that bridge the known-unknown gap in genomes and metagenomes. We showcase our approach by exploring 415,971,742 genes predicted from 1,749 metagenomes and 28,941 bacterial and archaeal genomes. We quantify the extent of the unknown fraction, its diversity, and its relevance across multiple biomes. Furthermore, we provide a collection of 283,874 lineage-specific genes of unknown function for Cand. Patescibacteria, being a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data.

microbiology↗

bioRxiv2020

Evaluating the impacts of microbial activity and species interactions on passive bioremediation of a coastal acid sulfate soil (CASS) ecosystem

Microbial iron and sulfate reduction are the primary drivers of coastal acid sulfate soil (CASS) passive bioremediation schemes. Microbial sulfate reduction is the limiting step for pyrite formation, a desirable endpoint for CASS remediation. Little is known, however, about the impacts of microbial activity or species interaction on long-term iron and sulfur cycling in CASS ecosystems. Using a combination of molecular biology, geochemical speciation and artificial intelligence-powered computational modeling, we deduced from microbial activity patterns (RNA-based) and geochemical measurements a best-fit equation for predicting biogeochemical pyrite formation in a model CASS ecosystem. In addition to the time-dependent activities of key sulfate-reducing prokaryotic taxa (e.g. Desulfobacteraceae), this equation required methylotrophs (Methylobacteriaceae) and bacterial predators (Bacteriovorax) for best-fitting, suggesting that specific microbial interactions exert meaningful influences on CASS bioremediation efficiency. Our findings confirmed that CASS microorganisms act as an assemblage in response to rewetting by tidewater. Accurate predictions of long-term CASS bioremediation efficiency require modelling of complex and interdependent relationships between geochemical speciation and microbial activity.HighlightsCoastal acid sulfate soil (CASS) is a global environmental issue.Microbial activity can be modelled quantitatively to predict CASS remediation.Sulfate-reducing prokaryotes (SRP) play a key role in CASS remediation.Predation on SRP with cultured representatives occurred during early wet-up.The above mechanism leads to increased activity among uncultured SRP.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2020

A simple method for isolating high-quality RNA from kenaf containing high polysaccharide and polyphenol contents

The isolation of high-quality RNA from kenaf is crucial for genetic and molecular biology studies. However, high levels of polysaccharide and polyphenol compounds in kenaf tissues could irreversibly bind to and coprecipitate with RNA, which complicates RNA extraction. In the present study, we proposed a simplified, time-saving and low-cost extraction method for isolating high quantities of high-quality RNA from several different kenaf tissues. RNA quality was measured for yield and purity, and the proposed protocol yielded high quantities of RNA (10.1-12.9 μg/g·FW). Spectrophotometric analysis showed that A260/280 ratios of RNA samples were in the range of 2.11 to 2.13, and A260/230 ratios were in the range of 2.04-2.24, indicating that the RNA samples were free of polyphenols, polysaccharides, and protein contaminants after isolation. The method of RNA extraction presented here was superior to the conventional CTAB method in terms of RNA isolation efficiency and was more sample-adaptable and cost-effective than commercial kits. Furthermore, to confirm downstream amenability, the high-quality RNA obtained from this method was successfully used for RT-PCR, real-time RT-PCR and Northern blot analysis. We provide an efficient and low-cost method for extracting high quantities of high-quality RNA from plants that are rich in polyphenols and polysaccharides, and this method was also validated for the isolation of high-quality RNA from other plants.View Full Text

genetics↗

bioRxiv2020

Activation of STING due to COPI-deficiency

COPA syndrome is caused by loss-of-function mutations in the COP- subunit of coatomer protein complex I (COPI), which participates in retrograde vesicular trafficking of proteins from the Golgi to the endoplasmic reticulum (ER). Disease manifests early in life with arthritis, lung pathology, kidney dysfunction and systemic inflammation associated with NF-{kappa}B activation and type I interferon (IFN{beta}) production. Here, we generated in vitro models for COPA syndrome and interrogated inflammatory signalling pathways via a range of biochemical and molecular biological techniques. Results were confirmed with cell lines in which mutant COPA was overexpressed and with COPA syndrome patient PBMCs. We identified Stimulator of Interferon Genes (STING), as a driver of inflammation in COPA syndrome. Furthermore, we found that genetic deletion of COPG1, another COPI subunit protein, induced NF-{kappa}B and type I IFN pathways similar to COPA-deficiency. Finally, we demonstrate that in vitro, inflammation due to COPA syndrome mutations was ameliorated by treatment with the small molecule STING inhibitor H-151. Therefore, inflammation induced by deletion of COPI subunits in general suggests a link between retrograde trafficking and STING regulation, and this innate immune sensor represents a novel therapeutic target in COPA syndrome.

immunology↗

bioRxiv2020

Forensic analysis of Soil Microbiomes: Linking Evidence to a Geographic Location

Over the past two decades, advances in molecular biology have greatly expanded our understanding of microbiomes - the diverse assemblages of microorganisms that inhabit the human body as well as the world around us, and applications in microbiome science have become an active area of research. Differences in the diversity (i.e., richness) and composition of microbiomes has been found to be informative in varied areas of science, including human health, agronomy, and forensic science. Soil harbors microbiomes that vary based on many factors, including the geology of the soil (e.g., sand, silt, or clay), climate, and use of the soil. As a result, the microbiological composition of any two soil samples will never be exactly alike. This inherent variation between microbiomes of different locations has proven to be specific enough to be potentially useful in forensic investigations to associate a person or piece of evidence to a source site. In this study, a soil microbiome was extracted from the sock of a criminal suspect and compared to the microbiome of soil samples taken from locations traveled to by the suspect. The locations analyzed varied in their soil microbiome composition, and the microbiome profiled from the sock was found to be most similar to the location where the suspect was thought to have left the body of a murder victim. These results provide a case study illustrating that information contained in a soil microbiome may be applied to link evidence to the location where a crime took place, potentially serving as an investigative tool in law enforcement.

bioinformatics↗