Organization of Nucleic Acids and Proteins by Lipid Membranes

AbstractOne of the peculiar aspects of living systems is the production and conservation of energy. This aspect is provided by specialized organelles, such as the mitochondria and chloroplasts, in developed living organisms. In primordial systems lacking specialized enzymatic complexes the energy supply was probably bound to the generation and maintenance of an asymmetric distribution of charged molecules in compartmentalized systems. On the basis of experimental evidence, we suggest that lipophilic quinones were involved in the generation of this asymmetrical distribution of charges through vectorial redox reactions across lipid membranes.

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Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076640 ◽

1983 ◽

Vol 41 ◽

pp. 608-609

Author(s):

Neng-Bo He ◽

S.W. Hui

Keyword(s):

Lipid Bilayers ◽

Erythrocyte Membrane ◽

Lipid Membranes ◽

Surface Film ◽

Biological Membranes ◽

Electron Microscopic Observation ◽

Electron Microscopic ◽

Black Lipid Membranes ◽

Fine Mesh ◽

Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

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Electron Microscopy of Nucleic Acids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051529 ◽

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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Direct visualization of phase-separated domains in lipid membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082315 ◽

1978 ◽

Vol 36 (2) ◽

pp. 290-291

Author(s):

S. W. Hui ◽

T. P. Stewart

Keyword(s):

Lipid Membranes ◽

Structural Information ◽

Freeze Fracture ◽

Biological Molecules ◽

Vacuum Drying ◽

Direct Visualization ◽

X Ray Diffraction ◽

Electron Microscopic ◽

X Ray ◽

Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

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A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102845 ◽

1988 ◽

Vol 46 ◽

pp. 152-153 ◽

Cited By ~ 1

Author(s):

A. Engel ◽

A. Holzenburg ◽

K. Stauffer ◽

J. Rosenbusch ◽

U. Aebi

Keyword(s):

Membrane Proteins ◽

Flow Rate ◽

Lipid Membranes ◽

Functional Characterization ◽

Dimensional Structure ◽

Electron Crystallography ◽

Peltier Element ◽

Protein Ratio ◽

Precise Control ◽

3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

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mRNA localization by Electron microscopic in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086453 ◽

1991 ◽

Vol 49 ◽

pp. 430-431

Author(s):

J. A. Pollock ◽

M. Martone ◽

T. Deerinck ◽

M. H. Ellisman

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Nucleic Acids ◽

Recombinant Dna ◽

Light And Electron Microscopy ◽

Electron Microscopic ◽

High Voltage Electron Microscopy ◽

Functional Sites ◽

Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

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Nucleic Acid Molecules Prepared by Monolayer Techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094140 ◽

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%.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124215 ◽

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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