Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis

Recombinant expression systems for production of stabilised virus-like particles as next-generation polio vaccines

Polioviruses have caused crippling disease in humans for centuries, prior to the successful development of vaccines in the mid-1900’s, which dramatically reduced disease prevalence. Continued use of these vaccines, however, threatens ultimate disease eradication and achievement of a polio-free world. Virus-like particles (VLPs) that lack a viral genome represent a safer potential vaccine, although they require particle stabilization. Using our previously established genetic techniques to stabilize the structural capsid proteins, we demonstrate production of poliovirus VLPs of all three serotypes, from four different recombinant expression systems. We compare the antigenicity, thermostability and immunogenicity of these stabilized VLPs against the current inactivated polio vaccine, demonstrating equivalent or superior immunogenicity in female Wistar rats. Structural analyses of these recombinant VLPs provide a rational understanding of the stabilizing mutations and the role of potential excipients. Collectively, we have established these poliovirus stabilized VLPs as viable next-generation vaccine candidates for the future

RNA-protein interactions in the assembly of tobacco mosaic virus

Assembly of tobacco mosaic virus is initiated by the binding of a specific loop of the RNA into the central hole of the disk aggregate of protein subunits. Since the nucleation loop is located about five-sixths along the RNA molecule, subsequent elongation must be bidirectional. We have now measured the rates of elongation in the two directions by determining the lengths of RNA protected from nuclease digestion at different times and using either intact TMV rNA, or RNA with most of the longer tail removed. Comparison of the rates with the protein supplied as either a mixture of disks with A-protein (a mixture of less aggregated states) or just A-protein, shows that different mechanisms and protein aggregates are used for the most rapid growth. When disks are present, they add more rapidly along the longer RNA tail but do not appear to add directly on the shorter tail. In contrast, smaller aggregates (A-protein) can add at both ends of the rod, but do so more slowly. Mechanisms for these processes are discussed. Preliminary results on the binding of the specific hexanucleotide AAGAAG to the disk are given and compared with the known changes on binding nonspecific hexanucleotides or the trinucleotide AAG