The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.
Barotrauma—injury induced by changes in pressure—is a widespread challenge for successfully releasing fishes following capture. We used acoustic telemetry to examine the long-term post-release survival and behaviour of four rockfish species (genus Sebastes) suffering from barotrauma following capture using recreational fishing techniques. We placed particular emphasis on examining Cowcod (Sebastes levis) and Bocaccio (Sebastes paucispinis), two historically overfished species along the United States West Coast that serve as good model species representing different ecological lifestyles. We show that fish survival was species specific and that 40% of observed mortality occurred more than 48 h post release—a typical time period used in many short-term survivorship studies. Cowcod survival was correlated with fish length, sea surface temperature, and dissolved oxygen concentration at the mean depth of capture. Generalized additive mixed models of Cowcod and Bocaccio behaviour showed that surviving individuals were negatively affected by capture and barotrauma for at least 30 d post-release. Our findings demonstrate the need for extended observations to accurately quantify the mortality of fishes suffering from barotrauma and show how such data can be successfully implemented into fisheries management through engagement between managers, scientists, and the fishing community.
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