scholarly journals An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

2019 ◽  
Author(s):  
Wim J. de Jonge ◽  
Mariël Brok ◽  
Patrick Kemmeren ◽  
Frank C.P. Holstege
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Magnetic Beads ◽  
Cell Lysis ◽  
Growth Conditions ◽  
Cross Linking ◽  
Optimized Protocol ◽  
A Genome ◽  
Study Results ◽  
Optimal Mix ◽  
Mechanical Lysis

AbstractChromatin immunoprecipitation (ChIP) is a commonly used technique to investigate which parts of a genome are bound by a particular protein. The result of ChIP is often interpreted in a binary manner: bound or not bound. Due to this focus, ChIP protocols frequently lack the ability to quantitatively compare samples with each other, for example in a time series or under different growth conditions. Here, using the yeast S. cerevisiae transcription factors Cbf1, Abf1, Reb1, Mcm1 and Sum1, we optimized the five major steps of a commonly used ChIP protocol: cross-linking, quenching, cell lysis, fragmentation and immunoprecipitation. Quenching with glycine is inefficient and can lead to large degrees of variability, an issue that is resolved by using tris(hydroxymethyl)aminomethane (Tris). Another source of variability is degradation of the protein of interest during the procedure. Enzymatic cell lysis with zymolyase can lead to extensive protein degradation, which is greatly reduced by mechanical lysis through bead beating. Degradation also occurs during sonication of chromatin, affecting large proteins in particular. An optimal mix of protease inhibitors and cross-linking with a higher percentage of formaldehyde reduces the extent of this degradation. Finally we also show that the immunoprecipitation step itself can be greatly improved with magnetic beads and optimized incubation/washing steps. The study results in a highly optimized protocol, which is shorter, easier to perform and has a stronger, more reproducible signal with less background. This protocol is presented in detail. In addition, the results highlight the greatest sources of variability in many other protocols, showing which steps are important to focus on for reproducible and quantitatively comparable ChIP experiments.

scholarly journals CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A. W. de Kok ◽  
Roel A. L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

Mediator dynamics during heat shock in budding yeast

2021 ◽  
pp. gr.275750.121
Author(s):  
Debasish Sarkar ◽  
Z. Iris Zhu ◽  
Elisabeth R. Knoll ◽  
Emily Paul ◽  
David Landsman ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Polymerase Ii ◽  
Budding Yeast ◽  
Core Promoter ◽  
Growth Conditions ◽  
Promoter Regions ◽  
Pol Ii ◽  
A Genome ◽  
Wide Scale ◽  
Stable Association

The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In budding yeast (Saccharomyces cerevisiae), Mediator is recruited by activators and associates with core promoter regions, where it facilitates pre-initiation complex (PIC) assembly, only transiently prior to Pol II escape. Interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes this association. However, Mediator occupancy and dynamics have not been examined on a genome-wide scale in yeast grown in nonstandard conditions. Here we investigate Mediator occupancy following heat shock or CdCl2 exposure, with and without depletion of Kin28. We find that Pol II occupancy exhibits similar dependence on Mediator under normal and heat shock conditions. However, while Mediator association increases at many genes upon Kin28 depletion under standard growth conditions, little or no increase is observed at most genes upon heat shock, indicating a more stable association of Mediator after heat shock. Mediator remains associated upstream of the core promoter at genes repressed by heat shock or CdCl2 exposure whether or not Kin28 is depleted, suggesting that Mediator is recruited by activators but is unable to engage PIC components at these repressed targets. This persistent association is strongest at promoters that bind the HMGB family member Hmo1, and is reduced but not eliminated in hmo1∆ yeast. Finally, we show a reduced dependence on PIC components for Mediator occupancy at promoters after heat shock, further supporting altered dynamics or stronger engagement with activators under these conditions.

Functional Magnetite Nanoparticle: A Review on the Particles Lysis and Nucleic Acid Separation

2021 ◽  
Vol 33 ◽  
pp. 13-27
Author(s):  
Puspita Nurlilasari ◽  
Camellia Panatarani ◽  
Mia Miranti ◽  
Savira Ekawardhani ◽  
Ferry Faizal ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Magnetite Nanoparticles ◽  
Magnetic Beads ◽  
Functional Materials ◽  
Cell Lysis ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Coating Materials ◽  
Magnetite Nanoparticle ◽  
Separation Cell

The functional magnetite nanoparticles are one of the most important functional materials for nucleic acid separation. Cell lysis and magnetic separation are two essential steps involve in optimizing nucleic acid extraction using the magnetic beads method. Many coating materials, coupling agents, chemical cell lysis, and several methods have been proposed to produce the specific desired properties for nucleic acid extraction. The important properties, such as biocompatibility, stability, linking ability, hydrophobicity, and biodegradable, were considered. The appropriate coating material of magnetite core and coupling agent are necessary to give biomolecules a possibility to link with each other through chemical conjugation. In this review, progress in functional magnetite nanoparticles to optimize the high binding performance in nucleic acid extraction is discussed.

221 EPIGENETIC DYNAMICS OF PLURIPOTENCY GENES IN THE CONTEXT OF DIFFERENTIATION AND NUCLEAR REPROGRAMMING DETERMINED BY Q2ChIP, A QUICK AND QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION TECHNIQUE APPLICABLE TO SMALL CELL SAMPLES

2007 ◽  
Vol 19 (1) ◽  
pp. 227 ◽  
Author(s):  
J. A. Dahl ◽  
C. K. Taranger ◽  
P. Collas
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Regulation Of Gene Expression ◽  
Cell Extract ◽  
Proximal Promoter ◽  
Pcr Analysis ◽  
Cross Linking ◽  
Nuclear Reprogramming ◽  
Distal Enhancer ◽  
Developmentally Regulated

Interactions between proteins and DNA are essential for cellular functions such as genomic stability, DNA replication and repair, chromosome segregation, transcription, and epigenetic silencing of gene expression. Chromatin immunoprecipitation (ChIP) is a key technique for mapping histone modifications and transcription factor binding on DNA and thereby unraveling the role of epigenetics in the regulation of gene expression. Current ChIP protocols require extensive sample handling and large numbers of cells (5-10 million). primarily owing to ample loss of material during the procedure. We altered critical steps of conventional ChIP to develop a quick and quantitative (Q2) ChIP assay suitable for cell numbers 100- to 1000-fold lower than those required for conventional ChIP. Key modifications of the ChIP procedure include (i) formaldehyde DNA–protein cross-linking in suspended cells, (ii) cross-linking in the presence of 20 mM sodium butyrate to enhance specificity of precipitation of acetylated histones, (iii) transfer of washed precipitated immune complexes to a clean tube ('tube shift') to increase ChIP specificity by virtually eliminating nonspecifically bound chromatin, and (iv) combination of cross-link reversal, protein digestion, and DNA elution into a single 2-h step. We used Q2ChIP to monitor changes in 6 histone H3 modifications on the human developmentally regulated genes OCT4 (POU5F1), NANOG, and LMNA (lamin A) in the context of retinoic acid (RA)-mediated differentiation of embryonal carcinoma cells and upon reprogramming of kidney epithelial 293T cells to pluripotency in carcinoma cell extract (Taranger et al. 2005 Mol. Biol. Cell 16, 5719–5735). Real-time PCR analysis of precipitated DNA unravels an unexpected two-step heterochromatin assembly elicited by RA on the OCT4 proximal promoter, proximal enhancer, and distal enhancer, and on the NANOG promoter, whereby methylation of H3K9 and H3K27 is followed by H3K9 deacetylation. H3K4 di- and trimethylation remain relatively unaffected by RA treatment. In contrast, reprogramming of 293T cells in carcinoma extract promotes assembly of histone marks characteristic of transcriptional induction of OCT4 and NANOG, such as acetylation and demethylation of H3K9. The results argue toward ordered chromatin repackaging at developmentally regulated promoters upon differentiation or, conversely, nuclear reprogramming to pluripotency.

169 AN OPTIMIZED PROTOCOL FOR EXTRACTING RNA FROM SINGLE BOVINE OOCYTES AND BLASTOMERES

2008 ◽  
Vol 20 (1) ◽  
pp. 164 ◽  
Author(s):  
M. Boelhauve ◽  
T. Guengoer ◽  
K. Zitta ◽  
V. Zakhartchenko ◽  
E. Wolf
Keyword(s):  
Liquid Nitrogen ◽  
Reverse Transcription ◽  
Magnetic Beads ◽  
Isolation Method ◽  
Total Rna ◽  
Qpcr Analysis ◽  
Optimized Protocol ◽  
Single Blastomeres ◽  
Bovine Oocytes ◽  
Rna Concentration

Quantitative PCR (qPCR) analysis of gene expression in single bovine oocytes or blastomeres from early preimplantation embryos needs to meet optimized requirements for the isolation of the RNA, the reverse transcription reaction, and even the qPCR. Normally, large amounts of tissues are required for extracting RNA, but the RNA recovery per embryo is too low for a reliable detection of low expressed genes. Therefore an optimized isolation method is essential for obtaining sufficient RNA recoveries of (a) a group of embryos/oocytes (n = 10), (b) a single oocyte/embryo, or (c) single blastomeres without inhibition of the subsequent steps. For each experiment studied, the RNA was reverse-transcribed with ExtremeScript-OLS� (Omni Life Science, Inc., Raynham, MA, USA) following the manufacturer's instructions. The transcript levels were analyzed by the detection of the genes STAT3 and LEPR. First, we compared different isolation protocols from the literature for the isolation of oocyte RNA[TriZol� (Invitrogen, Carlsbad, CA, USA) and RNAPure™ (Peqlab Biotechnologie, Erlangen, Germany) for isolation of total RNA; magnetic beads and Absolutely RNA™ Nanoprep (Stratogene, La Jolla, CA, USA) for mRNA; n = 12 per protocol] by measurement of the total RNA concentration (TriZol and RNAPure) and qPCR analysis (all isolation techniques). The results showed that RNAPure provided the highest transcript numbers (100% RNAPure v. 78% TriZol, 25% Nanoprep, and 5% magnetic beads, respectively; P ≤ 0.001) and also the highest total RNA concentration (2.1 ng µL–1 v. TriZol 1.5 ng µL–1 total RNA per oocyte; P ≤ 0.05). In the second experiment, we analyzed the influence of a coprecipitant [glycogen, linear acrylamide, SeeDNA (Amersham Biosciences, Freiburg, Germany); n = 12] on the RNA recovery and the inhibition of the subsequent reverse transcription and PCR processes. In the third experiment, the collection/storage of single oocytes was compared [RNAlater� (Applied Biosystems, Darmstadt, Germany) or liquid nitrogen; n = 20]. The use of the coprecipitant linear acrylamide (100% v. 80% for glycogen and 58% for SeeDNA; P ≤ 0.05) and the storage in liquid nitrogen (100% v. 84% for RNAlater; P ≤ 0.001) showed the highest RNA recoveries without inhibition. Furthermore, we analyzed (with the now optimized protocol) single blastomeres derived from 8- (n = 6) and 16-cell (n = 6) IVF embryos by mechanical treatment. The aim of this experiment was to analyze the expression divergences in blastomeres from normal cultured embryos without synchronization and, further, how many single blastomeres per embryo are required to obtain a comparable expression level of all blastomeres. The results showed that nearly 50% of an embryo was required (at least 4 blastomeres from an 8-cell and 7 blastomeres from a 16-cell embryo). These findings are mainly caused by different cell cycle phases of the analyzed blastomeres. Further studies with synchronized blastomeres are in progress. In conclusion, the present study demonstrates an effective RNA isolation method for a reliable qPCR analysis of single blastomeres. This work was supported by grant of the Deutsche Forschungsgemeinschaft (DFG) (FOR 478/1).

scholarly journals EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

2020 ◽  
Author(s):  
Jeanine Rismondo ◽  
Lisa M. Schulz ◽  
Maria Yacoub ◽  
Ashima Wadhawan ◽  
Michael Hoppert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Abc Transporter ◽  
Cell Lysis ◽  
Growth Conditions ◽  
Cell Wall Integrity ◽  
Growth Defect ◽  
Cell Wall Biosynthesis ◽  
High Concentrations

Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity. IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

scholarly journals Data on antigen recognition hindrance by antibodies covalently immobilized to Protein G magnetic beads by dimethyl pimelimidate (DMP) cross-linking

Data in Brief ◽  
2019 ◽  
Vol 22 ◽  
pp. 516-521 ◽  
Author(s):  
Marcelle A. Caminha ◽  
Virginia Maria B. de Lorena ◽  
Wilson de Oliveira Júnior ◽  
Jonas Perales ◽  
Paulo C. Carvalho ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Antigen Recognition ◽  
Cross Linking ◽  
Protein G

scholarly journals A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

2020 ◽  
Vol 19 (7) ◽  
pp. 1161-1178 ◽  
Author(s):  
Andreas Linden ◽  
Markus Deckers ◽  
Iwan Parfentev ◽  
Ralf Pflanz ◽  
Bettina Homberg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Isotopic Labeling ◽  
Growth Conditions ◽  
Cross Linking ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Protein Protein Interactions ◽  
Copy Numbers ◽  
Transport Chain ◽  
Quantitative Changes

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

scholarly journals Structure and Complexity of a Bacterial Transcriptome

2009 ◽  
Vol 191 (10) ◽  
pp. 3203-3211 ◽  
Author(s):  
Karla D. Passalacqua ◽  
Anjana Varadarajan ◽  
Brian D. Ondov ◽  
David T. Okou ◽  
Michael E. Zwick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Cell ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
A Genome ◽  
Nucleotide Resolution ◽  
Bacterial Transcriptome

ABSTRACT Although gene expression has been studied in bacteria for decades, many aspects of the bacterial transcriptome remain poorly understood. Transcript structure, operon linkages, and information on absolute abundance all provide valuable insights into gene function and regulation, but none has ever been determined on a genome-wide scale for any bacterium. Indeed, these aspects of the prokaryotic transcriptome have been explored on a large scale in only a few instances, and consequently little is known about the absolute composition of the mRNA population within a bacterial cell. Here we report the use of a high-throughput sequencing-based approach in assembling the first comprehensive, single-nucleotide resolution view of a bacterial transcriptome. We sampled the Bacillus anthracis transcriptome under a variety of growth conditions and showed that the data provide an accurate and high-resolution map of transcript start sites and operon structure throughout the genome. Further, the sequence data identified previously nonannotated regions with significant transcriptional activity and enhanced the accuracy of existing genome annotations. Finally, our data provide estimates of absolute transcript abundance and suggest that there is significant transcriptional heterogeneity within a clonal, synchronized bacterial population. Overall, our results offer an unprecedented view of gene expression and regulation in a bacterial cell.

scholarly journals Recombination mapping of the Brazilian stingless bee Frieseomelitta varia confirms high recombination rates in social hymenoptera

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Prashant Waiker ◽  
Fabiano Carlos Pinto de Abreu ◽  
Danielle Luna-Lucena ◽  
Flávia Cristina Paula Freitas ◽  
Zilá Luz Paulino Simões ◽  
...  
Keyword(s):  
Linkage Map ◽  
Social Evolution ◽  
Stingless Bee ◽  
Recombination Rates ◽  
Social Hymenoptera ◽  
Social Species ◽  
A Genome ◽  
Study Results ◽  
Worker Caste ◽  
Genomic Recombination

Abstract Background Meiotic recombination is a fundamental genetic process that shuffles allele combinations and promotes accurate segregation of chromosomes. Analyses of the ubiquitous variation of recombination rates within and across species suggest that recombination is evolving adaptively. All studied insects with advanced eusociality have shown exceptionally high recombination rates, which may represent a prominent case of adaptive evolution of recombination. However, our understanding of the relationship between social evolution and recombination rates is incomplete, partly due to lacking empirical data. Here, we present a linkage map of the monandrous, advanced eusocial Brazilian stingless bee, Frieseomelitta varia, providing the first recombination analysis in the diverse Meliponini (Hymenoptera, Apidae). Results Our linkage map includes 1417 markers in 19 linkage groups. This map spans approximately 2580 centimorgans, and comparisons to the physical genome assembly indicate that it covers more than 75 % of the 275 Megabasepairs (Mbp) F. varia genome. Thus, our study results in a genome-wide recombination rate estimate of 9.3–12.5 centimorgan per Mbp. This value is higher than estimates from nonsocial insects and comparable to other highly social species, although it does not support our prediction that monandry and strong queen-worker caste divergence of F. varia lead to even higher recombination rates than other advanced eusocial species. Conclusions Our study expands the association between elevated recombination and sociality in the order Hymenoptera and strengthens the support for the hypothesis that advanced social evolution in hymenopteran insects invariably selects for high genomic recombination rates.

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