RESUMEN
Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Rhabdoviridae/enzimología , Rhabdoviridae/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Baculoviridae/genética , Investigación Biomédica/organización & administración , Investigación Biomédica/tendencias , Enzimas/genética , Escherichia coli/genética , Unión Europea , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcripción Genética , Proteínas no Estructurales Virales/genéticaRESUMEN
The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
Asunto(s)
Células Eucariotas/metabolismo , Proteómica/métodos , Animales , Baculoviridae/genética , Células Cultivadas , Clonación Molecular , Expresión Génica , Glicosilación , Selenometionina , Levaduras/metabolismoRESUMEN
The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.
Asunto(s)
ADN/genética , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/química , Sondas de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/genética , ARN/química , ARN Bacteriano/química , ARN Bacteriano/genéticaRESUMEN
The folding of three- and four-way DNA junctions is often assessed by comparing the electrophoretic mobility of restriction enzyme fragments, using the long-short arm assay. We have compared the mobility of synthetic three-way junctions that contain identical branch point sequences, but different restriction sites in the arms. We show that the mobility of fragments is affected by the sequence of the overhanging ends. In general, GC-rich overhangs produce fragments with anomalous mobilities. These anomalies can be prevented by treating the cleaved junctions with mung bean endonuclease, elevating the electrophoresis temperature or using blunt cleaving restriction endonucleases.