Moving boundary electrophoresis on agarose gel of plant viruses and polystyrene microspheres (original) (raw)
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Electrophoresis, 1987
Abbreviations: ACES, 2-I(2-amino-2-oxoethyl)-aminol ethanesulfonic acid; CHAPS, 3-1(3-cholamidopropyI)-dimethylammoniol-I-propane sulfonate; Ferguson plot, Plot of log(mobi1ity) or log vs. %T; HEM, N-Phydroyethylmorpholine; HCRSV, hibiscus chlorotic ringspot virus; PFBV, pelargonium flowerbreak virus; Polybead. commercial name for polystyrene microsphere; %T, gel concentration(% w/v): TCV, turnip crinkle virus. S. J. Hayes L231 has come to our attention which uses polystyrene sulfate latex particles of 138 and 206 nm radius as size standards in 2-dimensional agarose gel eletrophoresis. 0173-0835/87/0606-026 I $02.50/0 0 VCH Verlagsgesellschaft mhH, D-6940 Weinheim, 1987 262 E. Gornbocz et al.
Electrophoresis, 1987
ABSTRACT Two procedures of computer simulation of the electrophoretic migration of a particle through agarose gel are described which allow for: (a) characterization of gel fiber dimensions as a function of gel concentration (gel standardization), (b) determination of particle radius and the dynamics of apparent particle compressibility during passage through the standardized gel, and (c) estimation of the net charge density of a particle by calculating its mobility at 0 % gel concentration. The common model underlying these simulations is based on the extended Ogston theory which probabilistically describes the migration of a particle through a random network of inert and non-flexible fibers in terms of a “random space walk”. The first procedure, applicable to relatively rigid particles such as bacteriophages, standardizes the gel fiber on the basis of mobility values (cm/s)/(V/cm) at several gel concentrations of a single, or several, bacteriophages of known radius. Mobilities of an unknown bacteriophage are then used to simulate its physical properties. The second method, applicable to relatively non-rigid particles such as plant viruses, uses 7 polystyrene particles of known radius to standardize the gel fiber, followed by simulation of virus properties on the basis of their mobilities at several gel concentrations. The techniques described are most appropriate for deriving physical properties of particles from their nonlinear plots of log (mobility) vs. gel concentration (Ferguson plots). They have the virtue of yielding the properties of native, hydrated gel fibers and particles.
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
Electrophoresis, 1988
Accurately standardized commercial polystyrene sulfate particles in agarose gel electrophoresis yield linear Ferguson plots at pH 7.4 over a gel concentration range up to0.9 % agarose which do not exhibit any significant sigmoidal curveelements, using either a discontinuous buffer system or a continuous buffer. Ferguson plots of these standard-sized particles were evaluated using alternatively a linear or convex model, by means of a newly developed set ofprograms (to be used in conjunction with program M-LAB) which (i) is sufficiently user-friendly to allow .for quantitative agarose gel electrophoresis of subcellular-sized spherical particles based on their convex Ferguson plots with the same operational simplicity previously available for linear Ferguson plots only; (ii) simultaneously and interactively analyzes the Ferguson plots of all particles under consideration on the basis of an extended Ogston model. 0 VCH Verlagsgesellschaft mbH, 0-6940 Weinheim, 1988 0173-0835/880606-0243%02.50/0 E. Hahn et al. Electrophoresis 1988,9,243-255 E. Hahn et a/.
Capillary electrophoresis of biological particles: Viruses, bacteria, and eukaryotic cells
ELECTROPHORESIS, 2004
A review about the application of electrophoretic methods in the capillary format for the investigation of large biological assemblies like viruses, bacteria, yeast or entire mammalian cells is given. These entities are of a size ranging between some nanometers and tens of micrometers. They can form colloidal solutions or dispersions and move under the influence of an electric field. They are separated by zone electrophoresis according to their different electrophoretic velocity, and characterized by the electrophoretic mobility, which is easily determinable in free solution in capillaries or in other microdevices. As the charge of these particles, when being amphoteric, is pH-dependent, isoelectric focusing can also be carried out and the capillary format is increasingly being employed for their separation and determination of pI values. Furthermore, interactions with ligands can be assessed by various modes of affinity capillary electrophoresis. Capillary zone electrophoresis has thus become a valuable tool for investigation of large macromolecular assemblies in the field of biochemistry, clinical chemistry, toxicology, and nutrition chemistry amongst many others.
The Journal of Physical Chemistry, 1994
Previous results on the electrophoretic mobility of aqueous suspensions of polystyrene latices at low ionic strengths are controversial. We have reexamined and extended mobility measurements as a function ofpolyion concentration down to salt concentrations smaller than ns = 10-6 M, where the polyions are liquid-like ordered due to screened Coulomb forces. A significant increase in mobility is found below ns 10-4 M. To discuss mobilities as a function of small ion concentration, for the first time electrophoretic mobility and electric conductivity data are combined to estimate salt content. Static light scattering is used to support the latter results and to characterize the structure of the suspensions. The weak polyion concentration dependence and the increase in mobility are discussed in the light of recently published calculations.
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.
Cell-to-cell movement of plant viruses
Archives of Virology, 1993
Cell-to-cell movement is a crucial step in plant virus infection. In many viruses, the movement function is secured by specific virus-encoded proteins. Amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. This superfamily combines proteins of viruses belonging to all principal groups of positive-strand RNA viruses, as well as singlestranded DNA containing geminiviruses, double-stranded DNA-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand RNA genomes with two ambisense segments. In several groups of positive-strand RNA viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative RNA helicase. A distinct type of movement proteins with very high content of proline is found in tymoviruses. It is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. Recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. Limited sequence similarities were observed between i) movement proteins of dianthoviruses and the MIP family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and M 1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. It is hypothesized that all movement proteins 240 A.R. Mushegian and E. V. Koonin of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules.