Polystyrene latex particles as size standards in quantitative agarose gel electrophoresis: Application to three plant viruses (original) (raw)
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Moving boundary electrophoresis on agarose gel of plant viruses and polystyrene microspheres
Electrophoresis, 1987
Three plant viruses: turnip crinkle (TCV), hibiscus chlorotic ringspot (HCRSV) and pelargonium flowerbreak (PFBV), and polystyrene size standards with radii of 22.4-59.4 nm can be stacked within trailing and leading ion net mobilities of 0.059 to 0.273 (relative to Na+). Stacking was carried out at pH 6.50,0.03 M ionic strength, 50 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, at a gel concentration of 0.4 %, in agarose gel electrophoresis conducted at 1.2 mA/cm2 of gel. Unstacking occurs under the same conditions at gel concentrations ranging from 0.5 to 1.1 % agarose, while it can be brought about between 0.1 and 0.7 % agarose when the pH is raised to 7.27, corresponding to a front moving boundary with a trailing ion net mobility of 0.216 (relative to Na+). Ferguson plots of viruses and polystyrene particles in the discontinuous buffer system are curvilinear and comparable to those obtained in a continuous buffer at pH 6.50 of the same composition and operative pH as that of the resolving phase of the discontinuous buffer. Particle radii and net charge values can be obtained from the non-linear Ferguson plot in the discontinuous buffer system by previously reported methods of computer simulation, but this Ferguson plot presents a more limited data base than that in the continuous buffer since it excludes gel concentrations which yield relative mobility (Rf) values of 1 .O. Since computer simulation provides the range of gel concentrations in which both thefiberradiusandlength [ 11, aswellasthesizeoftheparticle [2],remainconstantfor a particular preparation of agarose, a simplified alternative method of evaluating particle sizes exists. Within that specific gel concentration range, the linear segment of the Ferguson plot can be used to compute particle sizes by an operationally convenient, albeit approximative, method, using the same PAGE-PACK programs of D. Rodbard which are commonly used in the size determination of macromolecules by polyacrylamide gel electrophoresis. The two methods of particle size determination, based on either the entire non-linear Ferguson plot or on its linear segment in the appropriate gel concentration range, yield similar results (average deviation 12 %). The radii of three plant viruses are dependent to different degrees on the presence of Ca++ in the electrophoretic system. Values obtained in the presence of Ca++ are comparable to those found by electron microscopy.
Some Observations on the Structure of the Filamentous Particles of Several Plant Viruses
Journal of General Virology, 1968
Several plant viruses with filamentous particles ranging in modal lengths from 0"48/z to 1.25 # were negatively stained with uranyl formate, examined in the electron microscope, and the electron micrographs analysed in various ways. The particles of all the viruses were helically constructed with a basic pitch of 33 to 37 ~, (mean 34-~), but could be separated into groups by other features of their particles. Various measurements of the particles of five of the viruses suggest that there were IO to I4 subunits in each turn of the basic helix of their particles. All plant viruses with elongated particles seem to fall into one of two groups; those with modal lengths of 0"3/z or less seem rigid and have a basic helix of pitch 23 to 25 .~, and those with longer particles are filamentous and have a basic helix of pitch 33 to 37 ~,-Io7
Intervirology, 1986
The amino acid (AA) contents of the coat proteins of 134 plant viruses and strains were classified by principal components analysis. The virus groupings that were obtained correlated well with the classification of Mutthews. The relationships of each virus were dependent on the number of AA residues (axis 1) and on the percentage composition of each AA in the proteins (axes 2-4). The classification indicated which data were anomalous and needed confirmation. There seemed to be more anomalies in estimates of protein size than of protein composition. Tremaine and Goldsack [l] attempted, without success, to determine if there was a relationship between the amino acid composition (AAC) of the coat proteins (CPs) of the particles of plant viruses and the shapes of those particles. Tremaine aizdArgyle[2], using an agglomerative method of sorting strategy and the Euclidean distance metric, could not correlate the AAC of the CPs of plant viruses with groupings based on other classifications [3-51. Gibbs [6] chose the same criterion in an
Purification and electron microscopy of potato leafroll virus
Virology, 1969
A procedure for purification of potato leafroll virus (PLRV) from its plant host was improved. An extract from diseased Physalis $oridana plants was emulsified with an n-butanol-chloroform mixture. The virus in the aqueous phase was concentrated by centrifugation, the pellet was resuspended in 0.01 M phosphate buffer and subjected to emulsification with fluorocarbon (Daifron S-3). The virus, concentrated by additional centrifugation from the aqueous phase, was fractionated in a sucrose densitygradient column. When the final preparation was suspended in 0.01 M phosphate buffer at pH 6.0 and negatively stained by 2oJ, PTA at pH 5.5, the resulting suspension contained uniform particles 25 rnp in diameter (side by side) and with more or less hexagonal profiles. High infectivity was associated with the zone containing these particles. Preparations obtained from healthy plants and virus-free aphids by the same purification procedure did not contain these particles. Examination of ultrathin sections of the infected P. JEoridana and Datura stramonium plants by electron microscopy indicated that the virus occurred in some of the phloem cells. When PLRV particles were measured in crystalline array in ultrathin sections the size obtained (23 rnp diameters) agreed approximately with that of the purified virus particles.
Enzyme-assisted immune detection of plant virus proteins electroblotted onto nitrocellulose paper
Journal of Virological Methods, 1982
A technique for the detection of plant virus coat proteins in plant sap is described. The method entails the electroblotting of sodium dodecyi sulphate-polyacrylamide gel electrophoresis-fractionated plant extracts onto nitrocellulose paper, probing the paper with virus-specific rabbit antisera, and indirect detection of virus proteins with horseradish peroxidase-conjugated goat anti-rabbit globulins. The sensitivity and specificity of the technique were tested using brome mosaic and barley stripe mosaic viruses. As little as 1 ng per track of virus protein was detectable, either as pure virus or when mixed with plant sap. Distant serological relationships were detected amongst tobamoviruses, and amongst the bromoviruses, with single antisera. The uses of the technique in probing capsid configuration in a presumed aphid picornavirus, and in routine diagnostic practice, are described.
Journal of General Virology, 1989
The distribution of parsnip yellow fleck virus particle antigen in infected cells of Nicotiana clevelandii or Spinacia oleracea was examined by immunogold labelling of ultrathin sections. Best results were obtained by pretreating sections with Decon 75 followed by long incubation times on antiserum (16 h) and gold probe (6 h). The cytoplasmic inclusions induced by infection have three main components: accumulations of 20 to 30 nm diameter tubules, granular bodies and amorphous matrix material. Much gold label was located over the areas of amorphous matrix material whereas the other components of the inclusions were not labelled. Specific but less dense labelling was observed over virus-induced cell wall outgrowths, and overother areas of cell wall and some nuclei in infected cells. Virus-like particles found in 45 nm diameter tubules within the cell wall outgrowths were not labelled, perhaps because they were inaccessible to the antibody. The results indicate that large amounts of virus particle antigen are present in cells. However, the number of recognizable virus particles was considerably less than expected from the amount of virus extracted from leaves.