scholarly journals VISUALIZATION OF DEOXYRIBONUCLEIC ACID MOLECULES BY PROTEIN FILM ADSORPTION AND TANTALUM-TUNGSTEN SHADOWING

1974 ◽  
Vol 22 (9) ◽  
pp. 845-855 ◽  
Author(s):  
REINHARD ABERMANN ◽  
MIRIAM M. SALPETER
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Cytochrome C ◽  
Deoxyribonucleic Acid ◽  
Limiting Factors ◽  
Dna Molecules ◽  
Two Stage ◽  
Protein Film ◽  
Dna Strands

Preparatory procedures and high resolution shadowing of deoxyribonucleic acid (DNA) molecules for electron microscopy are described. They involve a two-stage mounting of the DNA on cytochrome c monolayers and shadowing with tantalum-tungsten. Conditions are determined that produce a smooth substrate and reproducible binding of the DNA strands in a randomly coiled manner without obvious stretching artifacts. When limiting factors such as excess buffer salts are eliminated, the DNA strands have the over-all dimensions expected from the naked molecule.

High Resolution Shadowing of DNA

1972 ◽  
Vol 30 ◽  
pp. 310-311
Author(s):  
R. Abermann ◽  
M. M. Salpeter
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Cytochrome C ◽  
Test Specimen ◽  
Electron Beam Evaporation ◽  
Absolute Ethanol ◽  
Dna Molecules ◽  
Step Process ◽  
Carbon Coated ◽  
Resolution Level

High resolution shadowing by electron beam evaporation was used to visualize DNA molecules. Its applicability to the study of macromolecules at this resolution level was previously demonstrated on monomeric and dimeric tRNA particles.To utilize the potential of the high resolution shadowing for DNA, a mounting procedure was employed which provided a smooth substrate to which the naked molecules could be bound. This consisted of a two step process, modified from Kleinschmidt Lang, and Harford and Beer. When a 50μl droplet of cytochrome c (20 μg/ml in 0.2M Na acetate at pH 5) was allowed to stand on teflon for 20 minutes, a monolayer of protein formed on its surface. The film was picked up onto carbon coated grids, washed in ethanol and air dried. As test specimen, we used DNA from SV4O. The protein coated grids were floated for 30 minutes on buffer droplets containing 0.1-0.5 μg DNA/ml 10-3SSC and the molecules were bound to the protein monolayers by diffusion. Grids were then washed successively in 50% and absolute ethanol and air dried.

Electron Microscopy of Mitochondrial DNA From Rat Brain

1972 ◽  
Vol 30 ◽  
pp. 188-189
Author(s):  
R. Paul Chowdhury ◽  
D. N. Misra ◽  
P. Sadhukhan
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Rat Brain ◽  
Brain Tissue ◽  
Heart Muscle ◽  
Deoxyribonucleic Acid ◽  
Flight Muscle ◽  
Albino Rats ◽  
Structural Form ◽  
Dna Molecules

The presence of deoxyribonucleic acid (DJNA) in the mitochondria of cells and tissues of vertebrates, invertebrates and plants is now well-established. The conformation of DNA has been extensively studied from highly purified preparations of mitochondria from liver, heart muscle and flight muscle and rarely from brain tissue. The present work has been conducted to study the structural form and length of DNA molecules of mitochondria from brain of albino rats.

scholarly journals The effects of deoxyribonucleic acid secondary structure on tertiary structure

1976 ◽  
Vol 159 (3) ◽  
pp. 615-620 ◽  
Author(s):  
A M Campbell
Keyword(s):  
Ionic Strength ◽  
Secondary Structure ◽  
Deoxyribonucleic Acid ◽  
Chromosome Structure ◽  
Tertiary Structure ◽  
Limiting Factors ◽  
Branch Points ◽  
Linear Dna ◽  
Dna Strands ◽  
Molecular Dimensions

The secondary structure of supercoiled DNA was varied by changes in ionic strength. For I = 0.075-0.4 the structure remained in the previously established branched form with only minor alterations in molecular dimensions. In 4M-NaCl, which induces linear DNA to change its secondary structure to the C structure and brings about an increase in the superhelix density of the molecule, no extra branches were observed on the molecules. The limiting factors that dictate supercoil structure seem to be the number and position of potential branch points and the proximity with which the two intertwining DNA strands can approach each other on the arms of the branches. This value is close to 10nm under the conditions described, and is 14-15nm at I = 0.2. It is suggested that such values should be borne in mind when models of chromosome structure are being constructed.

A Preparation of High Resolution Standards for Electron Microscopy. Part II

1971 ◽  
Vol 29 ◽  
pp. 160-161
Author(s):  
Akira Tanaka ◽  
David F. Harling
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Carbon Black ◽  
Single Crystal ◽  
Dark Field ◽  
Graphitized Carbon Black ◽  
Graphitized Carbon ◽  
Dark Field Imaging ◽  
Crystal Films ◽  
Micro Holes

In the previous paper, the author reported on a technique for preparing vapor-deposited single crystal films as high resolution standards for electron microscopy. The present paper is intended to describe the preparation of several high resolution standards for dark field microscopy and also to mention some results obtained from these studies. Three preparations were used initially: 1.) Graphitized carbon black, 2.) Epitaxially grown particles of different metals prepared by vapor deposition, and 3.) Particles grown epitaxially on the edge of micro-holes formed in a gold single crystal film.The authors successfully obtained dark field micrographs demonstrating the 3.4Å lattice spacing of graphitized carbon black and the Au single crystal (111) lattice of 2.35Å. The latter spacing is especially suitable for dark field imaging because of its preparation, as in 3.), above. After the deposited film of Au (001) orientation is prepared at 400°C the substrate temperature is raised, resulting in the formation of many square micro-holes caused by partial evaporation of the Au film.

Structural analysis of the surface-layer protein of spirillum serpens by high-resolution electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 738-739
Author(s):  
W. H. Wu ◽  
R. M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Structural Analysis ◽  
High Resolution ◽  
Low Dose ◽  
High Resolution Electron Microscopy ◽  
Optical Diffraction ◽  
Surface Layer Protein ◽  
Morphological Unit ◽  
Resolution Electron

Spirillum serpens possesses a surface layer protein which exhibits a regular hexagonal packing of the morphological subunits. A morphological model of the structure of the protein has been proposed at a resolution of about 25 Å, in which the morphological unit might be described as having the appearance of a flared-out, hollow cylinder with six ÅspokesÅ at the flared end. In order to understand the detailed association of the macromolecules, it is necessary to do a high resolution structural analysis. Large, single layered arrays of the surface layer protein have been obtained for this purpose by means of extensive heating in high CaCl2, a procedure derived from that of Buckmire and Murray. Low dose, low temperature electron microscopy has been applied to the large arrays.As a first step, the samples were negatively stained with neutralized phosphotungstic acid, and the specimens were imaged at 40,000 magnification by use of a high resolution cold stage on a JE0L 100B. Low dose images were recorded with exposures of 7-9 electrons/Å2. The micrographs obtained (Fig. 1) were examined by use of optical diffraction (Fig. 2) to tell what areas were especially well ordered.

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

High resolution surface structure of foot-and-mouth disease virus

1978 ◽  
Vol 36 (2) ◽  
pp. 376-377
Author(s):  
S. S. Breese ◽  
H. L. Bachrach
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Surface Structure ◽  
Disease Virus ◽  
Potassium Phosphate ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Physical Measurements ◽  
Mouth Disease Virus ◽  
Foot And Mouth

Models for the structure of foot-and-mouth disease virus (FMDV) have been proposed from chemical and physical measurements (Brown, et al., 1970; Talbot and Brown, 1972; Strohmaier and Adam, 1976) and from rotational image-enhancement electron microscopy (Breese, et al., 1965). In this report we examine the surface structure of FMDV particles by high resolution electron microscopy and compare it with that of particles in which the outermost capsid protein VP3 (ca. 30, 000 daltons) has been split into smaller segments, two of which VP3a and VP3b have molecular weights of about 15, 000 daltons (Bachrach, et al., 1975).Highly purified and concentrated type A12, strain 119 FMDV (5 mg/ml) was prepared as previously described (Bachrach, et al., 1964) and stored at 4°C in 0. 2 M KC1-0. 5 M potassium phosphate buffer at pH 7. 5. For electron microscopy, 1. 0 ml samples of purified virus and trypsin-treated virus were dialyzed at 4°C against 0. 2 M NH4OAC at pH 7. 3, deposited onto carbonized formvar-coated copper screens and stained with phosphotungstic acid, pH 7. 3.

Electron Microscopy of Nucleic Acids

1974 ◽  
Vol 32 ◽  
pp. 302-303
Author(s):  
Norman Davidson
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acids ◽  
Basic Protein ◽  
Molecular Biologist ◽  
Topological Properties ◽  
Protein Film ◽  
Polynucleotide Chain

The basic protein film technique for mounting nucleic acids for electron microscopy has proven to be a general and powerful tool for the working molecular biologist in characterizing different nucleic acids. It i s possible to measure molecular lengths of duplex and single-stranded DNAs and RNAs. In particular, it is thus possible to as certain whether or not the nucleic acids extracted from a particular source are or are not homogeneous in length. The topological properties of the polynucleotide chain (linear or circular, relaxed or supercoiled circles, interlocked circles, etc. ) can also be as certained.

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