The Identification of the Amino Acid Acceptor Terminus of tRNAPheyeast

1976 ◽  
Vol 34 ◽  
pp. 330-331
Author(s):  
A.P. Korn ◽  
F.P. Ottensmeyer
Keyword(s):  
Heavy Atom ◽  
Dark Field ◽  
Biological Macromolecules ◽  
Reaction Conditions ◽  
Heavy Atoms ◽  
Structural Details ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Amino Acid Acceptor

Employing the technique of beam tilt dark field electron microscopy we have been able to visualize in biological macromolecules structural details down to as much as 5 Å in size (1). In addition, images of individual heavy atoms have been obtained. These two achievements open the door to a new field of structure determination of proteins and nucleic acids: if a group of heavy atoms can be reacted specifically with a key position in the molecule, that position in the electron micrograph will be recognized by virtue of the presence of a cluster of heavy atom spots in that region. Requirements for the heavy atom reagent are 1) that it react specifically, 2) that the reaction conditions be sufficiently mild that the molecule is in its “native” conformation, and 3) that the presence of the heavy atom reagents not perturb the conformation of the molecule, at least to the extent that it can be detected by the technique.

The Structure of Macromolecules at 5 Angstroms

1975 ◽  
Vol 33 ◽  
pp. 14-15
Author(s):  
F. P. Ottensmeyer ◽  
R.F. Whiting ◽  
A.P. Korn
Keyword(s):  
Electron Microscopy ◽  
Heavy Atom ◽  
Chromatic Aberration ◽  
Dark Field ◽  
Resolving Power ◽  
Radiation Doses ◽  
Heavy Atoms ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Electron Microscopes

Most modern electron microscopes can boast a resolving power of about 3 A. Nevertheless, virtually all images of biological specimens display but a 20-30 A resolution, limited by the coarseness of the virtually obligatory heavy atom stain or shadow required for contrast and visibility. The loss in visibility incurred by the omission of heavy atoms can be overcome by the use of dark field electron microscopy. Moreover, for macromolecules, chromatic aberration, which limits the resolving power of the technique in the case of thick specimens, is not a problem. The sole remaining question then is whether the exquisitely fragile specimen, now unsupported and unprotected by heavy atom stains or shadows, can withstand the increasingly enormous radiation doses required to obtain the higher resolutions.

Studies of a Model DNA Sequence by Dark Field Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 384-385
Author(s):  
R.F. Whiting ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Dna Sequence ◽  
Dark Field ◽  
Heavy Atoms ◽  
Transmission Electron ◽  
Thin Carbon ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Small Model

Single heavy atoms in a variety of small model molecules have been managed by dark field transmission electron microscopy. The image intenses and geometry have been shown to be consistent with theoretical expectations. The use of this system for sequencing requires a demonstration theory accurate sequence can be derived for a small well-defined nucleic species. We have prepared and stained a homogeneous fragment of bacteria DNA. The molecule was a large pyrimidine oligomer anc b 5en sequenced biochemically. The sequence is shown in Fig. 1(a). Thymidine residues were marked with Beer's OsO4-cyanide stain. The refined, purified product was applied to pre-treated thin carbon specimen forms for electron microscopy.

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.

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