Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains

1991 ◽  
Vol 99 (3) ◽  
pp. 503-513
Author(s):  
A.V. Lichtenstein ◽  
M.M. Zaboikin ◽  
N.I. Sjakste ◽  
R.P. Alechina
Keyword(s):  
Protein Interactions ◽  
Dna Sequences ◽  
Attachment Site ◽  
Chromatin Domain ◽  
Dna Fragments ◽  
S1 Nuclease ◽  
Novel Approach ◽  
Attachment Sites ◽  
Dna Protein Interactions

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the ‘DNA adherence’ classes DNA-0, DNA-I and DNA-II, respectively. This ‘protein image’ DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.

scholarly journals Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli

2017 ◽  
Author(s):  
Andrey Shur ◽  
Richard M. Murray
Keyword(s):  
Dna Sequences ◽  
Dna Recombination ◽  
Attachment Site ◽  
Memory Systems ◽  
Rna Sequences ◽  
Guide Rna ◽  
Future Goals ◽  
Attachment Sites ◽  
Synthetic Cell ◽  
Specific Pair

AbstractSerine integrases are bacteriophage proteins responsible for integrating the phage genome into that of the host. Synthetic biologists have co-opted these proteins into useful tools for permanent DNA logic, utilizing their specific DNA recombination abilities to build synthetic cell differentiation and genetic memory systems. Each integrase has a specific pair of DNA sequences (attP/attB sites) that it recombines, but multiple identical sites can result in unpredictable recombination. We have developed a way to control integrase activity on identical attP/attB sites by using catalytically dead Cas9 (dCas9) as a programmable binding protein that can compete with integrase for binding to specific attachment sites. Utilizing a plasmid that contains two identical Bxb1 attP sites, integration can be repressed up to 8 fold at either one of the two attP sites when guide RNA and dCas9 are present. Guide RNA sequences that bind specifically to attB, or either of two attP sites, have been developed. Future goals are to utilize this technology to construct larger and more complex integrase logic circuits.

scholarly journals Reconfiguring primase DNA-recognition sequences by using a data-driven approach

2020 ◽  
Author(s):  
Adam Soffer ◽  
Morya Ifrach ◽  
Stefan Ilic ◽  
Ariel Afek ◽  
Dan Vilenchik ◽  
...  
Keyword(s):  
Machine Learning ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Metabolic Pathways ◽  
Dna Recognition ◽  
Model System ◽  
Data Driven ◽  
Specific Interactions ◽  
Data Driven Approach ◽  
Dna Protein Interactions

AbstractDNA–protein interactions are essential in all aspects of every living cell. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is challenging but important, since it may contribute to finding the means to regulate metabolic pathways involving DNA–protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA–protein interactions. Effective binding of T7 primase to its specific DNA recognition se-quences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.

DESIGNED SELF-ORGANIZATION FOR MOLECULAR OPTOELECTRONIC SENSORS

2007 ◽  
Vol 17 (02) ◽  
pp. 311-326 ◽  
Author(s):  
Michael Norton
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Terahertz Spectroscopy ◽  
Attachment Site ◽  
Surface Immobilization ◽  
Surface Attachment ◽  
One Dimensional ◽  
Synthetic Dna ◽  
Attachment Sites ◽  
Sensitivity And Selectivity

The convergence of terahertz spectroscopy and single molecule experimentation offers significant promise of enhancement in sensitivity and selectivity in molecular recognition, identification and quantitation germane to military and security applications. This paper provides a brief overview of the constraints set by single molecule recognition systems and reports the results of experiments which address fundamental barriers to the integration of large, patterned bio-compatible molecular opto-electronic systems with silicon based microelectronic systems. Central to this thrust is an approach involving sequential epitaxy on surface bound single stranded DNA one-dimensional substrates. The challenge of producing highly structured macromolecular substrates, which are necessary in order to implement molecular nanolithography, has been addressed experimentally by combining “designer” synthetic DNA with biosynthetically derived plasmid components. By design, these one dimensional templates are composed of domains which contain sites which are recognized, and therefore addressable by either complementary DNA sequences and/or selected enzymes. Such design is necessary in order to access the nominal 2 nm linewidth potential resolution of nanolithography on these one-dimensional substrates. The recognition and binding properties of DNA ensure that the lithographic process is intrinsically self-organizing, and therefore self-aligning, a necessity for assembly processes at the requisite resolution. Another requirement of this molecular epitaxy approach is that the substrate must be immobilized. The challenge of robust surface immobilization is being addressed via the production of the equivalent of molecular tube sockets. In this application, multi-valent core-shell fluorescent quantum dots provide a mechanism to prepare surface attachment sites with a pre-determined 1:1 attachment site : substrate (DNA) molecule ratio.

scholarly journals Identification of Campylobacter jejuni ATCC 43431-Specific Genes by Whole Microbial Genome Comparisons

2004 ◽  
Vol 186 (14) ◽  
pp. 4781-4795 ◽  
Author(s):  
Frédéric Poly ◽  
Deborah Threadgill ◽  
Alain Stintzi
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Dna Fragments ◽  
Helicobacter Hepaticus ◽  
Type Iv ◽  
Novel Approach ◽  
The Mean ◽  
Reading Frames

ABSTRACT This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.

scholarly journals Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.

1995 ◽  
Vol 130 (6) ◽  
pp. 1239-1249 ◽  
Author(s):  
H Yokota ◽  
G van den Engh ◽  
J E Hearst ◽  
R K Sachs ◽  
B J Trask
Keyword(s):  
Random Walk ◽  
Dna Sequences ◽  
Large Scale ◽  
Loop Model ◽  
Interphase Nuclei ◽  
Attachment Sites ◽  
The Relationship ◽  
Made In ◽  
Random Walk Path

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.

Scaffold attachments within the human genome

1997 ◽  
Vol 110 (21) ◽  
pp. 2673-2682 ◽  
Author(s):  
J.M. Craig ◽  
S. Boyle ◽  
P. Perry ◽  
W.A. Bickmore
Keyword(s):  
Dna Sequences ◽  
Constitutive Heterochromatin ◽  
In Situ Hybridisation ◽  
Fluorescence In Situ Hybridisation ◽  
Loop Size ◽  
Inactive X Chromosome ◽  
Attachment Sites ◽  
The Matrix ◽  
Eukaryotic Chromatin

It is generally agreed that, above the level of the 30 nm fibre, eukaryotic chromatin is constrained into loops, but there is disagreement about the nature of the substructure that serves to anchor loops and the DNA sequences that act as the attachment sites. This problem may stem from the very different methods that all purport to separate loop and attached DNAs. We have tested ideas about how the genome is arranged into loops by analysing the average loop size over different cytologically resolvable regions of human chromosomes using fluorescence in situ hybridisation with loop and attached DNA fractions. Variations in average loop size, along and between chromosomes, measurable at this level of resolution were small but significant and were dependent on the extraction method. This emphasises the fundamental differences between the nuclear substructure probed by different protocols. DNA attached to the nuclear ‘scaffold’ or ‘matrix’ hybridises preferentially to gene-poor regions of the genome (G-bands). Conversely, fractions attached to the nuclear ‘skeleton’ hybridise preferentially to gene-rich R-bands and sites of high levels of transcription. The inactive X chromosome has a deficit of associations with the nuclear skeleton but not with the matrix or scaffold. A large excess of attached sequences is found at some sites of constitutive heterochromatin, but not at centromeres.

Biolayer Interferometry for DNA-Protein Interactions v1

2021 ◽  
Author(s):  
John K Barrows ◽  
Michael Van Dyke
Keyword(s):  
Protein Interactions ◽  
Dna Sequences ◽  
Dna Probes ◽  
Binding Kinetics ◽  
Biolayer Interferometry ◽  
Optical Wavelength ◽  
Interaction Dynamics ◽  
Kinetics Of ◽  
Substrate Kinetics ◽  
Dna Protein Interactions

Biolayer interferometry (BLI) is a widely utilized technique for determining the interaction dynamics between macromolecules. Most BLI instruments, such as the Octet RED96e used throughout this protocol, are completely automated and detect changes in the interference pattern of white light reflected off a biosensor tip. Biosensors are initially loaded with a stationary macromolecule, then introduced into a solution containing macromolecules of interest. Binding to the stationary molecules creates a change in optical wavelength that is recorded by the instrument in real-time. The majority of published BLI experiments assess protein-protein (such as antibody-substrate kinetics) or protein-small molecule (such as drug discovery) interactions. However, a less-appreciated assay for BLI analysis is DNA-protein interactions. In our laboratory, we have shown the practicality of using biotinylated-DNA probes to determine the binding kinetics of transcription factors to specific DNA sequences. The following protocol describes these steps, including the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis through GraphPad Prism.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Tail labelled oligonucleotide probes for the detection of DNA-protein interactions

2011 ◽  
Author(s):  
Hana Pivoňková ◽  
Kateřina Němcová ◽  
Petra Horáková ◽  
Luděk Havran ◽  
Hana Macíčková-Cahová ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Oligonucleotide Probes ◽  
Dna Protein Interactions
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