Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology.

Fluorescent-dye-conjugated oligonucleotides were used to classify 14 Fibrobacter strains by fluorescence microscopy. On the basis of partial 16S rRNA sequences of six Fibrobacter strains, four hybridization probes were designed to discriminate between the species Fibrobacter succinogenes and Fibrobacter intestinalis and to identify F. succinogenes subsp. succinogenes. After in situ hybridization to whole cells of the six sequenced strains, epifluorescence microscopy confirmed probe specificity. The four probes were then used to make presumptive species and subspecies assignments of eight additional Fibrobacter strains not previously characterized by comparative sequencing. These assignments were confirmed by comparative sequencing of the 16S rRNA target regions from the additional organisms. Single-mismatch discrimination between certain probe and nontarget sequences was demonstrated, and fluorescent intensity was shown to be enhanced by hybridization to multiple probes of the same specificity. The direct detection of F. intestinalis in mouse cecum samples demonstrated the application of this technique to the characterization of complex natural samples.  

Initial interaction of herpes simplex virus with cells is binding to heparan sulfate.

We have shown that cell surface heparan sulfate serves as the initial receptor for both serotypes of herpes simplex virus (HSV). We found that virions could bind to heparin, a related glycosaminoglycan, and that heparin blocked virus adsorption. Agents known to bind to cell surface heparan sulfate blocked viral adsorption and infection. Enzymatic digestion of cell surface heparan sulfate but not of dermatan sulfate or chondroitin sulfate concomitantly reduced the binding of virus to the cells and rendered the cells resistant to infection. Although cell surface heparan sulfate was required for infection by HSV types 1 and 2, the two serotypes may bind to heparan sulfate with different affinities or may recognize different structural features of heparan sulfate. Consistent with their broad host ranges, the two HSV serotypes use as primary receptors ubiquitous cell surface components known to participate in interactions with the extracellular matrix and with other cell surfaces.  

Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.  

Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.