scholarly journals Bioinformatics Analysis v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Immunoprecipitation, Decross-linking, and DNA Extraction v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Determine the Size of Sonicated Samples and the DNA Concentration v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Cell Lysis and Sonication v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromosome Segregation ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Cross-Linking and Cell Harvesting v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Cell Harvesting ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals ChIP-seq Library preparation v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Library Preparation ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals AMPure Purification Protocol v1

2020 ◽  
Author(s):  
Vasso Makrantoni ◽  
Daniel Robertson ◽  
Adele L. Marston
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Dna Recombination ◽  
Yeast Cells ◽  
Biological Processes ◽  
Technological Advancement ◽  
Entire Genome ◽  
Protein Dna Interactions ◽  
Generation Sequencing ◽  
Purification Protocol

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

scholarly journals Budding yeast complete DNA replication after chromosome segregation begins

2018 ◽  
Author(s):  
Tsvetomira Ivanova ◽  
Michael Maier ◽  
Alsu Missarova ◽  
Céline Ziegler-Birling ◽  
Lucas B. Carey ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Normal Growth ◽  
Population Level ◽  
Mitotic Exit ◽  
Yeast Cells ◽  
Cyclin Dependent Kinase ◽  
Temporal Separation ◽  
Entire Genome ◽  
Number Variation

AbstractTo faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in a substantial fraction of unperturbed yeast cells, DNA replication finishes during anaphase, late in mitosis. High cyclin-Cdk activity inhibits replication in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA replication to finish at difficult-to-replicate regions. Replication during late mitosis correlates with elevated mutation rates, including copy number variation. Thus, yeast cells temporally overlap replication and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.One Sentence SummaryCompletion of DNA replication is coupled to downregulation of Cyclin-Dependent Kinase during mitotic exit.

Chromatin Immunoprecipitation of GFP-Tagged PML-Rara Coupled to High-Throughput Next Generation Sequencing.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1276-1276
Author(s):  
Nicole R. Grieselhuber ◽  
Jahangheer S. Shaik ◽  
Li-Wei Chang ◽  
Sean McGrath ◽  
Lukas D. Wartman ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Binding ◽  
Fusion Protein ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Next Generation ◽  
High Confidence ◽  
Altered Expression ◽  
Dna Binding Sites ◽  
Generation Sequencing

Abstract Abstract 1276 Poster Board I-298 The PML-RARA fusion protein produced by the t(15;17) translocation is found in acute promyelocytic leukemia (APL) and acts as an aberrant transcription factor with oncogenic properties. To define the high affinity DNA binding sites of PML-RARA, we developed a novel system based upon chromatin immunoprecipitation of eGFP tagged PML-RARA (PR), coupled to next generation sequencing. Chromatin isolated from flow sorted, GFP+ PR9 cells (isolated 24 hours after electroporation with eGFP-PR) was immunoprecipitated with a highly specific anti-GFP monoclonal antibody, and massively parallel sequencing was performed using single-end read libraries generated from both input DNA and immunoprecipitated DNA. The sequenced reads were mapped to the reference human genome using the Burrows-Wheeler Alignment tool (BWA). Using the SamTools package, alignment files were then filtered to retain only reads with phred quality scores greater than 30. Finally, Model Based Analysis of ChIP-Seq (MACS) was used to obtain the predicted binding sites (approximately 13,000 for each replicate using a p value cutoff of 0.00001). To ensure the reproducibility of called peaks, we limited our analysis to 701 sites that occurred in replicate sequencing runs of the libraries, within a tolerance of ±50 bp at the peak. Visual inspection of graphically plotted peaks further filtered this list to 421 high quality sites. Using microarray expression data for 14 APL patients and 5 flow sorted, normal promyelocyte samples, we selected neighboring genes with at least a 3-fold difference between APL and promyelocyte expression. This step yielded a list of 82 neighboring genes whose expression may be altered by PML-RARA binding to adjacent DNA. While PML-RARA is often considered a transcriptional repressor, 68% of these genes were overexpressed in APL, suggesting that PML-RARA may also function as a transcriptional activator. Furthermore, 51 genes were dysregulated in the mCG-PML-RARA murine model of APL, 16 genes demonstrated expression changes following induction of PML-RARA expression in PR9 cells, and 12 genes had altered expression in both the PR9 and murine models. Collectively, these results demonstrate an association between the high confidence putative PR binding sites and gene expression changes. We next used the sequences found within the best 89 binding sites to define 6 potential in vivo consensus sites of PML-RARA using the CONSENSUS program. Three of these predicted sites were present in greater than 40% of the 421 high confidence sites. Two of these common motifs resemble the motifs found in retinoic acid response elements (RAREs), but have less stringent conservation at the 5' end while retaining the 3' TCA sequence. Taken together, our results suggest that PML-RARA has an extended repertoire of genomic DNA binding sites compared to wild-type RARA, reflecting novel gain-of-function properties of the fusion protein. Binding of some of these sites appears to have direct consequences for the expression of several tightly linked genes, which may themselves be involved in transcriptional regulatory networks that contribute to APL pathogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Matthew J. Bush ◽  
Maureen J. Bibb ◽  
Govind Chandra ◽  
Kim C. Findlay ◽  
Mark J. Buttner
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Regulatory Networks ◽  
Liquid Culture ◽  
Biological Processes ◽  
Streptomyces Venezuelae ◽  
Content Type ◽  
Master Regulators ◽  
Aerial Growth ◽  
Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

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