Translation of the 5.7 kb luteovirus genome is controlled by the 3’ untranslated region (UTR). Base pairing between regions of the 3’ UTR and sequences kilobases upstream is required for cap-independent translation and ribosomal frameshifting needed to synthesize the viral replicase. Luteoviruses produce subgenomic RNAs, which can serve as mRNA, but one sgRNA also regulates translation initiation in trans. As on all viruses, the 3’ and 5’ ends contain structures that are presumed to facilitate RNA synthesis. This review describes the structures and interactions of Barley yellow dwarf virus RNA that facilitate the complex interplay between the above events and result in a successful virus infection. We also present surprising results on the apparent lack of need for some subgenomic RNAs for the virus to infect cells or whole plants. In summary, the UTRs of luteoviruses are highly complex entities that control and fine-tune many key events of the virus replication cycle.

SummaryBeta 2-glycoprotein I plays a pivotal role in the binding of antiphospholipid antibodies to phospholipid in patients with antiphospholipid syndrome. In this study the nature of the epitopes on beta 2-glycoprotein I (β2-GPI) recognised by sera from antiphospholipid syndrome (APS) patients (n = 15) was investigated and compared to rabbit polyclonal and mouse monoclonal anti-β2-GPI antibodies. β2-GPI was only recognised when bound to a high affinity binding support. The antigenic epitope on β2-GPI recognised by all APS patients was also dependent on disulphide bond integrity. Digestion of β2-GPI with elastase rapidly destroyed the epitope(s) on β2-GPI recognised by antibodies in 91% of APS patients. The main cleavage occurred at tryptophan316-lysine317 in the fifth domain. Digestion with staphylococcal V8 protease resulted in a 50% reduction in antibody binding in 81% of patients and the cleavage sites mainly involved the first domain of the molecule. There was considerable variability in the recognition of six different species of β2-GPI by serum from APS patients. The epitopes on β2-GPI bound by APS sera appear conformationally determined in all patients but are quite heterogeneous in the regions of β2-GPI that are recognised.

Protoplasts provide a convenient system for introduction of nucleic acids into plant cells. Protoplasts allow rapid assays of gene expression and virus replication, with advantages for plant biology similar to those of cultured animal cells for the study of animal systems. Traditionally, preparation and handling of protoplasts has been as much art as science, requiring a special touch by the user. The purpose of this unit is to lay out in clear detail all the methods and nuances involved in protoplast preparation using a robust, reliable system that does not require skills beyond those expected of an unspecialized molecular biologist. Because dicots and monocots differ in many biological properties, and because different procedures may work better for different plants, separate units in this chapter are devoted to protoplast preparation from dicots (Arabidopsis, tobacco; refer to UNITS 16D.1 & 16D.4) and from a monocot (oat). This unit describes methods for preparation and transfection by electroporation of protoplasts derived from an oat suspension culture.

The most common glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position of potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved by observing a correct mass-loss between a potential sugar substrate and its corresponding product using electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly-expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.

No abstract