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.

Dark-field electron spectroscopic imaging of aged microstructures in an aluminium lithium base alloy

1990 ◽  
Vol 48 (4) ◽  
pp. 444-445
Author(s):  
I. G. Solórzano ◽  
W. Probst
Keyword(s):  
Base Alloy ◽  
Spectroscopic Imaging ◽  
Chromatic Aberration ◽  
Dark Field ◽  
Objective Lens ◽  
Electron Spectroscopic Imaging ◽  
Bright Field ◽  
Field Mode ◽  
Dark Field Electron ◽  
Very High

The examination of microstructures make very high demands on the imaging quality and, therefore, on the instrumentation. In Al-Li base alloys it is of great interest to determine parameters such as size, distribution, morphology and coherency of precipitate phases as they dictate their mechanical behavior. In order to reveal morphological features with high quality the electron spectroscopic imaging (ESI) in dark field mode has shown to be quite a powerful technique.The ESI technique in the TEM is based on the possibility that accelerated electrons can be elastic and inelastically scattered by the sample atoms, as recently reviewed. The electron distribution in the transmitted and diffracted beams through a crystalline sample is such that both energy loss and elastic electrons will enter a typical objective aperture and thus contribute to both bright field and dark field images. The effect of the polyenergetic electrons is that the image is affected by chromatic aberration of the objective lens. In CTEM’s this effect is enhanced the lower the accelerating voltage and the thicker the sample.

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.

Utilizing Dark Field Electron Microscopy to Produce Lantern Slides

1974 ◽  
Vol 32 ◽  
pp. 116-117
Author(s):  
J. N. Meador ◽  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Electron Microscope ◽  
Electron Micrographs ◽  
Dark Field ◽  
Limiting Factor ◽  
Clinical Laboratories ◽  
Field Electron ◽  
Time Element ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Dark Field Micrographs

The electron microscope is being utilized more and more in clinical laboratories for pathologic diagnosis. One of the major problems in the utilization of the electron microscope for diagnostic purposes is the time element involved. Recent experimentation with rapid embedding has shown that this long phase of the process can be greatly shortened. In rush cases the making of projection slides can be eliminated by taking dark field electron micrographs which show up as a positive ready for use. The major limiting factor for use of dark field micrographs is resolution. However, for conference purposes electron micrographs are usually taken at 2.500X to 8.000X. At these low magnifications the resolution obtained is quite acceptable.

Protamine-DNA interaction

1978 ◽  
Vol 36 (2) ◽  
pp. 200-201
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Small Volume ◽  
Double Helix ◽  
Dna Interaction ◽  
Dark Field ◽  
Sperm Head ◽  
Nuclear Proteins ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Dna Double Helix ◽  
Positively Charged

Dark field electron microscopy has been used for the study of the structure of individual macromolecules with a resolution to at least the 5Å level. The use of this technique has been extended to the investigation of structure of interacting molecules, particularly the interaction between DNA and fish protamine, a class of basic nuclear proteins of molecular weight 4,000 daltons.Protamine, which is synthesized during spermatogenesis, binds to chromatin, displaces the somatic histones and wraps up the DNA to fit into the small volume of the sperm head. It has been proposed that protamine, existing as an extended polypeptide, winds around the minor groove of the DNA double helix, with protamine's positively-charged arginines lining up with the negatively-charged phosphates of DNA. However, viewing protamine as an extended protein is inconsistent with the results obtained in our laboratory.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Thin Pyrolytic Graphite Films for Electron Microscope Substrates

1971 ◽  
Vol 29 ◽  
pp. 226-227 ◽  
Author(s):  
George H. N. Riddle ◽  
Benjamin M. Siegel
Keyword(s):  
Vacuum Chamber ◽  
Pyrolytic Graphite ◽  
High Vacuum ◽  
Dark Field ◽  
Ultra High Vacuum ◽  
Graphite Substrate ◽  
Nickel Substrate ◽  
Routine Procedure ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A routine procedure for growing very thin graphite substrate films has been developed. The films are grown pyrolytically in an ultra-high vacuum chamber by exposing (111) epitaxial nickel films to carbon monoxide gas. The nickel serves as a catalyst for the disproportionation of CO through the reaction 2C0 → C + CO2. The nickel catalyst is prepared by evaporation onto artificial mica at 400°C and annealing for 1/2 hour at 600°C in vacuum. Exposure of the annealed nickel to 1 torr CO for 3 hours at 500°C results in the growth of very thin continuous graphite films. The graphite is stripped from its nickel substrate in acid and mounted on holey formvar support films for use as specimen substrates.The graphite films, self-supporting over formvar holes up to five microns in diameter, have been studied by bright and dark field electron microscopy, by electron diffraction, and have been shadowed to reveal their topography and thickness. The films consist of individual crystallites typically a micron across with their basal planes parallel to the surface but oriented in different, apparently random directions about the normal to the basal plane.

Characteristic Geometry and Diffraction Contrast of Dispersed ThO2 Crystals

1969 ◽  
Vol 27 ◽  
pp. 168-169
Author(s):  
R. J. Horylev ◽  
L. E. Murr
Keyword(s):  
Dark Field ◽  
Particle Orientation ◽  
Contrast Effects ◽  
Strain Fields ◽  
Diffraction Contrast ◽  
Dispersed Particles ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Particle Images ◽  
Diffraction Effects

Smith has shown by dark-field electron microscopy of extracted ThO2 particles from TD-nickel (2% ThO2) that they possess single crystal characteristics. It is generally assumed that these particle dispersions are incoherent. However, some diffraction effects associated with the particle images appeared to be similar to coherency strain fields. The present work will demonstrate conclusively that ThO2 dispersed particles in TD-nickel (2% ThO2) and TD-NiCr (2% ThO2, 20% Cr, Ni) are single crystals. Moreover, the diffraction contrast effects are extinction fringes. That is, these effects arise because of the particle orientation with respect to the electron beam and the extinction conditions for various operating reflections The particles are in fact incoherent.

Applications of Through-Focus Dark Field Image Shifts to Phase Identification

1975 ◽  
Vol 33 ◽  
pp. 178-179
Author(s):  
Prakash Rao
Keyword(s):  
Dark Field Image ◽  
Thin Foil ◽  
Dark Field ◽  
The Other ◽  
Stereo Image ◽  
Phase Identification ◽  
The Past ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Recent Extension

Image shifts in out-of-focus dark field images have been used in the past to determine, for example, epitaxial relationships in thin films. A recent extension of the use of dark field image shifts has been to out-of-focus images in conjunction with stereoviewing to produce an artificial stereo image effect. The technique, called through-focus dark field electron microscopy or 2-1/2D microscopy, basically involves obtaining two beam-tilted dark field images such that one is slightly over-focus and the other slightly under-focus, followed by examination of the two images through a conventional stereoviewer. The elevation differences so produced are usually unrelated to object positions in the thin foil and no specimen tilting is required.In order to produce this artificial stereo effect for the purpose of phase separation and identification, it is first necessary to select a region of the diffraction pattern containing more than just one discrete spot, with the objective aperture.

Structure and Interaction of Protamine by Dark Field Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 160-161
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Field Electron ◽  
Proposed Model ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Positively Charged

It has been shown for some time that it is possible to obtain images of small unstained proteins, with a resolution of approximately 5Å using dark field electron microscopy (1,2). Applying this technique, we have observed a uniformity in size and shape of the 2-dimensional images of pure specimens of fish protamines (salmon, herring (clupeine, Y-l) and rainbow trout (Salmo irideus)). On the basis of these images, a model for the 3-dimensional structure of the fish protamines has been proposed (2).The known amino acid sequences of fish protamines show stretches of positively charged arginines, separated by regions of neutral amino acids (3). The proposed model for protamine structure (2) consists of an irregular, right-handed helix with the segments of adjacent arginines forming the loops of the coil.

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