Nucleosome reconstruction by electron spectroscopic imaging of phosphorus

1984 ◽  
Vol 42 ◽  
pp. 168-169
Author(s):  
F.P. Ottensmeyer ◽  
G. Harauz
Keyword(s):  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Projection Algorithm ◽  
Dimensional Structure ◽  
Electron Spectroscopic Imaging ◽  
Base Pairs ◽  
Phosphorus Distribution ◽  
Nucleosome Core ◽  
Nucleosomal Dna ◽  
A New Technique

The three dimensional structure of a biological macromolecule or complex can be reconstructed from electron micrographs of different macromolecules or complexes lying in random orientation, provided that the relative orientations of individual particles can be determined (figure 1). In the case of the nucleosome core particle, a complex comprising 146 base pairs of DNA and 8 histone proteins, such orientational information can be obtained using a new technique called electron spectroscopic imaging which permits not only electron microscopy of the structure, but also the visualisation at high spatial resolution of the phosphorus distribution within the particle. Since phosphorus is a predominant constituent of DNA and not of protein, this elemental map represents an image of the nucleosomal DNA component. A comparison of a projection of a DNA supercoil model with images of the phosphorus distribution allowed us to orient individual distributions (figure 2). Using a direct convolution back-projection algorithm, the entire nucleosome containing both protein and DNA information was then reconstructed to a resolution of about 1.5 nm.

Three-dimensional imaging of nucleosomes from transcriptionally active genes using electron spectroscopic imaging

1996 ◽  
Vol 54 ◽  
pp. 54-55
Author(s):  
G. J. Czarnota ◽  
D. P. Bazett-Jones ◽  
F. P. Ottensmeyer
Keyword(s):  
Gene Expression ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Dimensional Structure ◽  
Crystallographic Structure ◽  
Electron Spectroscopic Imaging ◽  
Nucleosome Structure ◽  
Active Genes

The three-dimensional structure of the nucleosome was determined using particles purified from transcriptionally active genes in conjunction with electron spectroscopic imaging, and quaternion-assisted angular reconstitution procedures. The results reveal a configuration which is very different from the canonical compact crystallographic structure for this fundamental chromosome subunit, implying a structural disruption of the nucleosome with the activation of gene expression in accord with numerous physico-chemical observations.Previous analyses of nucleosomes purified from transcriptionally quiescent genes have indicated numerous structural states dependent on factors in vitro which modify charge based interactions in nucleoprotein complexes. Nucleosomes from transcriptionally active genes undergo chemical alterations in vivo which similarly modify charge based interactions. In order to investigate the effects of the gene expression associated chemical alterations on nucleosome structure, particles were purified from transcriptionally active genes using mercury affinity chromatography. These nucleosome particles are hyperacetylated with respect to particles from transcriptionally quiescent genes. Here additionally, sulphydryls normally buried within the protein core of the transcriptionally inactive particle are exposed to chemical modifying agents thus facilitating purification as described.

DNA organization in nucleosomes

1982 ◽  
Vol 60 (3) ◽  
pp. 364-370 ◽  
Author(s):  
D. P. Bazett-Jones ◽  
F. P. Ottensmeyer
Keyword(s):  
Spectroscopic Imaging ◽  
Dark Field ◽  
Electron Spectroscopic Imaging ◽  
Nucleosome Core ◽  
Dna Organization ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
A New Technique ◽  
Beaded Appearance ◽  
Active Genes

A new technique known as electron spectroscopic imaging has allowed the direct visualization of DNA within the nucleosomes of chromatin. The results presented here confirm the model which suggests that approximately two supercoil turns of DNA are wound about the nucleosome core. The structure of nucleosomes from putative transcriptionally active genes, fractionated by preferential sensitivity to DNAase II and solubility in 2 mM MgCl2, has been examined using both dark field electron microscopy and electron spectroscopic imaging. Oligomeric strands of nucleosomes in this fraction have a less distinct beaded appearance than those of bulk chromatin. The phosphorus distribution in this chromatin suggests that the DNA has a less recognizable organization, lacking a two-turn supercoil per subunit. The unique appearance of this fraction in 30 mM NaCl is reversibly changed to the classical beaded appearance when dialyzed into 0.4 M NaCl.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

Phosphorus distribution in perichromatin granules and surrounding nucleoplasm as visualized by electron spectroscopic imaging

1996 ◽  
Vol 87 (3) ◽  
pp. 171-177
Author(s):  
Gerardo H. Vázquez Nin ◽  
Sousan Abolhassani-Dadras ◽  
Olga M Echeverría ◽  
Viviane Boutinard Rouelle-Rossier ◽  
Stanislav Fakan
Keyword(s):  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Phosphorus Distribution ◽  
Perichromatin Granules

Imaging, representation and analysis of 3-dimensional biological structures by confocal microscopy

1988 ◽  
Vol 46 ◽  
pp. 996-997
Author(s):  
G.J. Brakenhoff ◽  
H.T.M. van der Voort
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Confocal Scanning Laser Microscopy ◽  
Optical Sectioning ◽  
Basic Aspect ◽  
3 Dimensional ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser ◽  
A New Technique

Confocal scanning laser microscopy is a new technique capable of visualizing the three-dimensional structure in suitable objects. As further explained in fig. 1 the basic aspect of this type of microscopy is that one and the same object point in the specimen is both optimally illuminated from a laser illuminated pinhole as well as optimally imaged on a detector pinhole. Due to the specific properties of this optical arrangement not only the lateral resolution is substantially improved, but also an excellent optical sectioning property comes available. The latter means that only information from a chosen plane in the specimen will contribute to the image, while the influence of the layers above and below is suppressed. The important advantage over elec- tronmicroscopy, apart from the speed of the technique, is that to make the 3-D information accessible in a specimen no mechanical sectioning of the material is necessary.

scholarly journals DNA information: from digital code to analogue structure

2012 ◽  
Vol 370 (1969) ◽  
pp. 2960-2986 ◽  
Author(s):  
A. A. Travers ◽  
G. Muskhelishvili ◽  
J. M. T. Thompson
Keyword(s):  
Dimensional Space ◽  
Double Helix ◽  
Three Dimensional ◽  
Base Pairs ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Regulatory Mode ◽  
Sequence Periodicity ◽  
Recent Developments ◽  
Dna Double Helix

The digital linear coding carried by the base pairs in the DNA double helix is now known to have an important component that acts by altering, along its length, the natural shape and stiffness of the molecule. In this way, one region of DNA is structurally distinguished from another, constituting an additional form of encoded information manifest in three-dimensional space. These shape and stiffness variations help in guiding and facilitating the DNA during its three-dimensional spatial interactions. Such interactions with itself allow communication between genes and enhanced wrapping and histone–octamer binding within the nucleosome core particle. Meanwhile, interactions with proteins can have a reduced entropic binding penalty owing to advantageous sequence-dependent bending anisotropy. Sequence periodicity within the DNA, giving a corresponding structural periodicity of shape and stiffness, also influences the supercoiling of the molecule, which, in turn, plays an important facilitating role. In effect, the super-helical density acts as an analogue regulatory mode in contrast to the more commonly acknowledged purely digital mode. Many of these ideas are still poorly understood, and represent a fundamental and outstanding biological question. This review gives an overview of very recent developments, and hopefully identifies promising future lines of enquiry.

scholarly journals 2-amino-1,3-benzothiazole-6-carboxamide Preferentially Binds the Tandem Mismatch Motif r(UY:GA)

2020 ◽  
Author(s):  
Andrew T. Chang ◽  
Lu Chen ◽  
Luo Song ◽  
Shuxing Zhang ◽  
Edward P. Nikonowicz
Keyword(s):  
Small Molecules ◽  
Dipolar Coupling ◽  
Residual Dipolar Coupling ◽  
Three Dimensional ◽  
Coupling Constants ◽  
Dimensional Structure ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Virtual Screen ◽  
Rna Hairpin

AbstractRNA helices are often punctuated with non-Watson-Crick features that can be the target of chemical compounds, but progress towards identifying small molecules specific for non-canonical elements has been slow. We have used a tandem UU:GA mismatch motif (5’-UG-3’:5’-AU-3’) embedded within the helix of an RNA hairpin as a model to identify compounds that bind the motif specifically. The three-dimensional structure of the RNA hairpin and its interaction with a small molecule compound identified through a virtual screen are presented. The G-A of the mismatch forms a sheared pair upon which the U-U base pair stacks. The hydrogen bond configuration of the U-U pair involves the O2 of the U adjacent to the G and the O4 of the U adjacent to the A. The G-A and U-U pairs are flanked by A-U and G-C base pairs, respectively, and the mismatch exhibits greater stability than when the motif is within the context of other flanking base pairs or when the 5’-3’ orientation of the G-A and U-U is swapped. Residual dipolar coupling constants were used to generate an ensemble of structures against which a virtual screen of 64,480 small molecules was performed to identify candidate compounds that the motif specifically binds. The tandem mismatch was found to be specific for one compound, 2-amino-1,3-benzothiazole-6-carboxamide, which binds with moderate affinity but extends the motif to include the flanking A-U and G-C base pairs. The finding that affinity for the UU:GA mismatch is flanking sequence dependent emphasizes the importance of motif context and potentially increases the number of small non-canonical features within RNA that can be specifically targeted by small molecules.

Magnetic Microscopy for 3D Structures: Use of the Simulation Approach for the Precise Localization of Deep Buried Weak Currents

2010 ◽  
Author(s):  
Fulvio Infante ◽  
Rodolphe Gomes ◽  
Philippe Perdu ◽  
Fabien Battistella ◽  
Sebastien Annereau ◽  
...  
Keyword(s):  
Magnetic Measurements ◽  
Three Dimensional ◽  
New Method ◽  
Dimensional Structure ◽  
Precise Localization ◽  
Simulation Approach ◽  
Three Dimensional Structure ◽  
Current Path ◽  
A New Technique ◽  
Failure Localization

Abstract With the innovations in packaging technologies which have taken place over the last decade, new assemblies often include an increasing number of dies inside a single package. This is exactly what was predicted by the More than Moore’s paradigm: as the integration of ICs increases, the heterogeneity of the devices found in a single package increases. As a result, the number of potential failures which can appear at assembly level has increased exponentially. At present, no technique has been able to precisely localize defects which are deep inside a complex package. For this reason, a new technique for failure localization for three-dimensional structures is needed. In this paper the technique proposed, based on the coupling of magnetic measurements and simulations, is applied to a three-dimensional structure to precisely localize the current path which is buried deep inside it. A new method, based on parameters fittings of magnetic simulations, is then applied in order to accurately evaluate the distance between the current and the sensor.

scholarly journals Probing Ribosomal RNA By Electron Spectroscopic Imaging and Three-Dimensional Reconstruction

1997 ◽  
Vol 5 (1) ◽  
pp. 10-11
Author(s):  
Daniel R. Beniac ◽  
Gregory J. Czarnota ◽  
Brenda L. Rutherford ◽  
F. Peter Ottensmeyer ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Elemental Distribution ◽  
Two Dimensional ◽  
Electron Spectroscopic Imaging ◽  
Ribosomal Subunits ◽  
Microanalytical Technique ◽  
Dimensional Reconstruction

The ribosome is the protein synthetic machinery in the cell. Knowledge of the structures of ribosomal RNA (rRNA) macromolecules in situ is essential to understanding their roles in ribosome mediated protein synthesis. We are using a microanalytical technique that identifies and maps elements directly, electron spectroscopic imaging, to determine the rRNA phosphorus distributions within Escherichia coli ribosomal subunits, and to combine the two-dimensional maps into a three-dimensional elemental distribution by iterative quaternion-assisted angular reconstitution of ribosomal particles at random orientations.

2. Genes as DNA

2014 ◽  
Author(s):  
Jonathan Slack
Keyword(s):  
Molecular Biology ◽  
Double Helix ◽  
Complex Structure ◽  
Genetic Material ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Human Beings ◽  
Base Pairs ◽  
Modern Molecular Biology ◽  
Complete Sequencing

After 1944, a remarkable set of discoveries established the overall shape of modern molecular biology and most famous of all was the discovery of the three dimensional structure of DNA: the famous double helix, which explained how the substance could act as the genetic material. ‘Genes as DNA’ describes the complex structure of genes and explains the terms ‘genome’ and ‘genomics’. In the 1980s and 1990s the complex mechanisms by which genes control embryonic development were discovered. The complete sequencing of a typical human genome was started in the late 1990s and achieved in 2003. It showed that the genome of human beings contains about three billion base pairs of DNA.

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