DNA Contour Length Measurements as a Tool for the Structural Analysis of DNA and Nucleoprotein Complexes

2011 ◽  
pp. 235-254
Author(s):  
Claudio Rivetti
Keyword(s):  
Structural Analysis ◽  
Contour Length ◽  
Length Measurements ◽  
Nucleoprotein Complexes

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%.

scholarly journals The C-terminal domain of ParB is critical for dynamic DNA binding and bridging interactions which condense the bacterial centromere

2017 ◽  
Author(s):  
Gemma L. M. Fisher ◽  
César L. Pastrana ◽  
Victoria A. Higman ◽  
Alan Koh ◽  
James A. Taylor ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Dna Binding ◽  
Dna Condensation ◽  
Dna Binding Domain ◽  
Dynamic Nature ◽  
Terminal Domain ◽  
Binding Interface ◽  
Nucleoprotein Complexes

SUMMARYThe ParB protein forms DNA bridging interactions aroundparSto form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring atparS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensationin vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formationin vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

Partial Denaturation Mapping of Phage T4 DNA at Low Temperature

1981 ◽  
Vol 36 (11-12) ◽  
pp. 980-987
Author(s):  
G. F. Grossi ◽  
M. F. Macchiato ◽  
G. Gialanella
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Computer Program ◽  
Contour Length ◽  
High Concentration ◽  
Experimental Conditions ◽  
Endonucleolytic Cleavage ◽  
Phage T4 ◽  
Length Measurements ◽  
Partial Denaturation

Abstract The partial-denaturation map of T4 DNA is obtained by using benzyldodecyldimethyl ammonium chloride in the presence of a high concentration of formamide. In this way suitable conditions for preparation of electron microscope specimens and partial denaturation within a temperature range low enough to minimize the endonucleolytic cleavage, are realized. It is found that, under our experimental conditions, the denaturation increase depends mainly on the appearance of new denaturation sites rather than on the lengthening of DNA segments already denatured. Because of the DNA circular permutation it is necessary to align the measured maps to obtain the denaturation pattern. This is done through a computer program and informations on the distribution of the regions with highest (A-T) content along the genome are obtained. The results of contour length measurements of λ and T4 DNA’s are also reported.

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.

Studies of Circular DNA Using a New Electron Microscopic Technique

1973 ◽  
Vol 31 ◽  
pp. 596-597
Author(s):  
C. N. Gordon
Keyword(s):  
Electron Microscopy ◽  
Aqueous Salt Solution ◽  
Contour Length ◽  
Cleavage Surface ◽  
Mica Substrate ◽  
Electron Microscopic ◽  
Replica Technique ◽  
Circular Dna ◽  
Transmission Electron ◽  
Electron Microscopic Technique

Gordon and Kleinschmidt have described a new preparative technique for visualizing DNA by electron microscopy. This procedure, which is a modification of Hall's “mica substrate technique”, consists of the following steps: (a) K+ ions on the cleavage surface of native mica are exchanged for Al3+ ions by ion exchange. (b) The mica, with Al3+ in the exchange sites on the surface, is placed in a dilute aqueous salt solution of DNA for several minutes; during this period DNA becomes adsorbed on the surface. (c) The mica with adsorbed DNA is removed from the DNA solution, rinsed, dried and visualized for transmission electron microscopy by Hall's platinum pre-shadow replica technique.In previous studies of circular DNA by this technique, most of the molecules seen were either broken to linears or extensively tangled; in general, it was not possible to obtain suitably large samples of open extended molecules for contour length measurements.

Existence of DNA in yeast microbodies

1978 ◽  
Vol 36 (2) ◽  
pp. 232-233
Author(s):  
Masako Osumi ◽  
Misuzu Nagano ◽  
Hiroko Kazama
Keyword(s):  
Catalase Activity ◽  
Buoyant Density ◽  
Model System ◽  
Contour Length ◽  
Native State ◽  
Normal Alkane ◽  
Remarkable Increase ◽  
Dna Molecule ◽  
Mitochondrial Dnas

We have found that microbodies appeared profusely together with a remarkable increase in catalase activity in normal alkane-grown cells of hydrocarbon-utilizing Candida yeasts, and that the microbodies multiplied by division in these cells. These features of Candida yeasts seem to provide a useful model system for studies on the biogenesis of the microbody. Subsequently, we have succeeded in isolation of Candida microbodies in an apparently native state, as judged biochemically and morphologically. The presence of DNA in the purified microbody fraction thus obtained was proved by the diphenylamine method. DNA molecule of about 15 urn in contour length was released from an isolated microbody. The physicochemical analyses of the microbody DNA revealed that its buoyant density differed from nuclear and mitochondrial DNAs. All these results lead us to the possibility that there is a novel type of DNA in microbodies.

RNA polymerase: Preparation and structural analysis of helical polymers

1984 ◽  
Vol 42 ◽  
pp. 152-153
Author(s):  
E. Loren Buhle ◽  
Pamela Rew ◽  
Ueli Aebi
Keyword(s):  
Structural Analysis ◽  
Rna Polymerase ◽  
Molecular Level ◽  
X Ray Diffraction ◽  
Helical Polymers ◽  
X Ray ◽  
E Coli ◽  
The Past ◽  
Ordered Arrays ◽  
Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

Electron Microscope Study of RNA's of Paramyxoviruses

1972 ◽  
Vol 30 ◽  
pp. 196-197
Author(s):  
Ruchama Baum ◽  
J.T. Seto
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Microscope Study ◽  
Sendai Virus ◽  
Sodium Dodecyl ◽  
Rna Molecules ◽  
Virus Particles ◽  
Molecular Weights ◽  
Length Measurements

The ribonucleic acid (RNA) of paramyxoviruses has been characterized by biochemical and physiochemical methods. However, paramyxovirus RNA molecules have not been studied by electron microscopy. The molecular weights of these single-stranded viral RNA molecules are not known as yet. Since electron microscopy has been found to be useful for the characterization of single-stranded RNA, this investigation was initiated to examine the morphology and length measurements of paramyxovirus RNA's.Sendai virus Z strain and Newcastle disease virus (NDV), Milano strain, were used. For these studies it was necessary to develop a method of extracting RNA molecules from purified virus particles. Highly purified Sendai virus was treated with pronase (300 μg/ml) at 37°C for 30 minutes and the RNA extracted by the sodium dodecyl sulfate (SDS)-phenol procedure.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

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