Observation of Fast Sedimenting, Replicating Bacteriophage T7 DNA by Negative Staining

1980 ◽  
Vol 38 ◽  
pp. 538-539
Author(s):  
P. Serwer
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Dna Packaging ◽  
Negative Staining ◽  
Bacteriophage T7 ◽  
Dna Complexes ◽  
Sufficient Detail ◽  
Protein Monolayer

To package the DNA of bacteriophage T7, a preformed, DNA-free capsid (capsid I) with an envelope thicker than the envelope of bacteriophage T7:(a) binds DNA, (b) converts to a capsid (capsid II) with a bacteriophage-like envelope prior to packaging DNA and (c) draws in DNA (1). During attempts to understand T7 DNA packaging, complexes of capsids with mature T7 DNA and complexes of capsids with longer than mature, linear T7 DNA have been isolated (2). Objects with capsid-like dimensions were observed on a fast sedimenting, replicating complex of T7 DNA (100S+ DNA) prepared for electron microscopy using a protein monolayer-shadowing technique (3). This procedure for preparation of specimens does not, however, reveal sufficient detail to rigorously identify an object as a capsid. To better visualize objects bound to 100S+ T7 DNA, this DNA has been prepared for electron microscopy using the aqueous technique for the negative staining of capsid-DNA complexes more recently described (4) (the DNA is coated with cytochrome c and is revealed in an extended configuration).

Nucleic Acid Molecules Prepared by Monolayer Techniques

1972 ◽  
Vol 30 ◽  
pp. 178-179
Author(s):  
Dimitrij Lang
Keyword(s):  
Electron Microscopy ◽  
Standard Deviation ◽  
Nucleic Acid ◽  
Cytochrome C ◽  
Nucleic Acids ◽  
Contour Length ◽  
Sample Standard Deviation ◽  
Molecular Weights ◽  
Length Measurements ◽  
Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

Electron Microscopy of Bacteriophage T7 Internal Head Proteins

1975 ◽  
Vol 33 ◽  
pp. 638-639
Author(s):  
P. Serwer
Keyword(s):  
Electron Microscopy ◽  
Bacteriophage T7 ◽  
Specimen Preparation ◽  
Dna Molecule ◽  
The Core ◽  
Internal Core ◽  
Phage T7 ◽  
T7 Phage ◽  
Preparation Techniques ◽  
Internal Head

The genome of bacteriophage T7 is a duplex DNA molecule packaged in a space whose volume has been measured to be 2.2 x the volume of the B form of T7 DNA. To help determine the mechanism for packaging this DNA, the configuration of proteins inside the phage head has been investigated by electron microscopy. A core which is roughly cylindrical in outline has been observed inside the head of phage T7 using three different specimen preparation techniques.When T7 phage are treated with glutaraldehyde, DNA is ejected from the head often revealing an internal core (dark arrows in Fig. 1). When both the core and tail are present in a particle, the core appears to be coaxial with the tail. Core-tail complexes sometimes dislodge from their normal location and appear attached to the outside of a phage head (light arrow in Fig. 1).

scholarly journals 1616. Mechanism of Thrombocytopenia Induced by Oxazolidinones Antibiotics (Linezolid, Tedizolid): Demonstration of Impairment of Megakaryocyte Differentiation From Human Hematopoietic Stem Cells associated with Mitochondrial Toxicity

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S801-S801
Author(s):  
Paul M Tulkens ◽  
Tamara V Milosevic ◽  
Gaëlle Vertenoeil ◽  
William Vainchenker ◽  
Stefan N Constantinescu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Stem Cells ◽  
Cytochrome C ◽  
Hematopoietic Stem Cells ◽  
Cytochrome C Oxidase ◽  
Panel Member ◽  
Human Model ◽  
Mitochondrial Toxicity ◽  
Review Panel ◽  
Hematopoietic Stem

Abstract Background Linezolid causes thrombocytopenia, which limits its use. In cell culture and in tissues from treated patients, linezolid impairs mitochondrial protein synthesis (due to structural similarities and common binding sites between bacterial and mitochondrial ribosomes). Recent studies have shown that mitochondria act as a key relay in the process leading from activation of the thrombopoietin receptor to megakaryocytes differentiation. Methods Validated ex-vivo human model of hematopoietic stem cells (HSC) differentiation for (i) measuring megakaryocytes, granulocyte-monocytes, and burst-forming unit-erythroids colony formation; (ii) differentiation into megakaryocytes (conversion of CD34+ into CD41+/CD42+ cells; morphology) and proplatelets formation, (iii) mitochondrial toxicity (electron microscopy; cytochrome c-oxidase activity [partly encoded by the mitochondrial genome]). Results We show that linezolid (and the recently approved tedizolid), both at concentrations corresponding to their human serum concentrations) inhibit the maturation of HSC into fully differentiated megakaryocytes (CD41 and CD42-positive cells) and the formation of proplatelets. Optic and Electron microscopy) showed an impairment of the formation of typical megakaryocytes (lack of large polylobulated nuclei and of intracellular demarcation membrane system [required for platelet formation]), together with disappearance of the internal structure of mitochondria. Biochemical studies showed a complete suppression of the activity of cytochrome c-oxidase (a key enzyme of the mitochondrial respiratory chain). Conclusion Our study provides for the first time insights in the mechanism of thrombocytopenia induced by linezolid and tedizolid, identifying mitochondria as their target and showing that the drugs will impair the differentiation of hematopoietic stem cells into mature platelets-releasing megakaryocytes. It illustrates how mitochondria dysfunction may play a key role in toxicology and diseases, while paving the way for rational approaches for the design and screening of less toxic derivatives for the benefit of future patients. Disclosures Paul M. Tulkens, MD, PhD, Bayer (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Menarini (Speaker’s Bureau)Merck (Advisor or Review Panel member, Speaker’s Bureau)Trius (now part of Merck) (Advisor or Review Panel member, Research Grant or Support) Françoise Van Bambeke, PharmD, PhD, Bayer (Speaker’s Bureau)

The fine structure and the protein composition of γ phage of Bacillus anthracis

1975 ◽  
Vol 21 (11) ◽  
pp. 1889-1892 ◽  
Author(s):  
Takashi Watanabe ◽  
Akinori Morimoto ◽  
Toshiro Shiomi
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Bacillus Anthracis ◽  
Protein Composition ◽  
Staining Technique ◽  
Negative Staining ◽  
Polyacrylamide Gels ◽  
Long Tail

The fine structure of γ phage of Bacillus anthracis was studied by electron microscopy with a negative-staining technique. The phage has a hexagonal head and a long tail without a sheath. By electrophoresis on polyacrylamide gels, the proteins of the phage particles are separate into 10 polypeptides with moleclar weights ranging from 140 000 to 12 000.

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