PubMed · 9760508
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J Wellink. 1998. Comovirus isolation and RNA extraction.. https://doi.org/10.1385/0-89603-385-6%3A205
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Viruses are attractive building blocks for nanoscale heterostructures, but little is understood about the physical principles governing their directed assembly. In situ force microscopy was used to investigate organization of Cowpea Mosaic Virus engineered to bind specifically and reversibly at nanoscale chemical templates with sub-30 nm features. Morphological evolution and assembly kinetics were measured as virus flux and inter-viral potential were varied. The resulting morphologies were similar to those of atomic-scale epitaxial systems, but the underlying thermodynamics was analogous to that of colloidal systems in confined geometries. The 1D templates biased the location of initial cluster formation, introduced asymmetric sticking probabilities, and drove 1D and 2D condensation at sub-critical volume fractions. The growth kinetics followed a t(1/2) law controlled by the slow diffusion of viruses. The ability of poly(ethylene glycol) (PEG) to induce the lateral expansion of virus clusters away from the 1D templates suggests a significant role for weak interactions.
A new technique for conducting site-directed mutagenesis was developed. This method allows the colour selection of mutants through the simultaneous activation or deactivation of the alpha-peptide of beta-galactosidase. Double-stranded DNA plasmids containing large inserts (at least 6.4 kbp in the present experiments) can be used as the mutational template. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. The utility of the method for the analysis of viral genomes was demonstrated by applying it to the mutagenesis of a full-length cDNA copy of RNA-1 of Cowpea mosaic virus (CPMV). Six single-site mutants were initially produced which gave a variety of phenotypes when inoculated on to plants. To confirm that the phenotypes were directly caused by the introduced mutations, a second round of mutagenesis was used to create revertants of two of the mutants. In both cases, the revertants had a wild-type phenotype, demonstrating that the original phenotype was, indeed, the result of the introduced mutation. Overall, the results show that the present technique is a powerful method for site-directed mutagenesis of large DNA fragments, such as whole viral genomes, for functional studies.
This paper reports experimental results on the dielectrophoretic (DEP) behaviour on two nonenveloped plant viruses of different geometrical shapes, namely Cow Pea Mosaic Virus (CPMV) and Tobacco Mosaic Virus (TMV). The DEP properties of carboxy-modified latex beads of the same size are also reported. The DEP properties of single particles were obtained from measurement of the frequency at which the DEP force on a particle goes to zero (the crossover frequency). The DEP behaviour of particle ensembles was also measured using image processing. The dielectric properties of the particles were evaluated from the DEP data. The surface conductance was found to be 0.3 nS for CPMV, 0.38 nS for TMV, and 0.52 nS for 27 nm diameter carboxy-latex beads. Data analysis has shown that the optimal condition for separation of TMV and CPMV is a low-conductivity suspending medium - below 1 mS/m.