Evolution of a biological thermocouple by adaptation of cytochrome c oxidase in a subterrestrial metazoan.

In this study we report a naturally evolved temperature-sensing electrical regulator in the cytochrome c oxidase of the Devil Worm, Halicephalobus mephisto. This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. We obtained the full mitochondrial genome sequence of this organism, and show through dN/dS analysis statistically robust evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of these positively-selected amino acid substitutions were localized in proximity to the H- and K-pathway proton channels of the complex. Surprisingly, the H. mephisto cytochrome c oxidase proton pump completely shuts down at low temperatures (20°C) leading to approximately a 4.8-fold reduction in the transmembrane proton gradient voltage (ΔΨm) compared to optimal temperature (37°C). Direct measurement of oxygen consumption found a corresponding 4.7-fold drop at 20°C compared to 37°C. Correspondingly, the lifecycle of H. mephisto takes four-fold longer at the low temperature compared to higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success in varying environmental temperatures. Our study shows that evolutionary innovation may remodel core metabolism to make it more accurately map onto environmental variation.

Stress Adapted Mollusca and Nematoda Exhibit Convergently Expanded Hsp70 and AIG1 Gene Families.

We recently sequenced the genome of the first subterrestrial metazoan, the nematode Halicephalobus mephisto. A central finding was a dramatic expansion of genes encoding avrRpt2 induced gene (AIG1), and 70 kDa heat shock (Hsp70) domains. While the role of Hsp70 in thermotolerance is well established, the contribution of AIG1 is much more poorly characterized, though in plants some members of this family are heat-induced. Hypothesizing that this dual domain expansion may constitute a general biosignature of thermal stress adaptation, here we examine a number of genomes, finding that expansion of both AIG1 and Hsp70 is common in bivalves. Phylogenetic analysis reveals that the bivalve-specific Hsp70 protein expansion groups with H. mephisto sequences. Our identification of the same gene expansions in bivalves and a nematode implies that this biosignature may be a general stress adaptation strategy for protostomes, particularly those organisms that cannot escape their stressful environments. We hypothesize that the two families play largely complementary mechanistic roles, with Hsp70 directly refolding heat-denatured proteins while AIG1 promotes cellular and organismal survival by inhibiting apoptosis.

Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen.

Flow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts the relative expression level of the therapeutic protein for each clone. This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96-well plate clone screening and elimination of unstable clones at an earlier stage in the development process. Furthermore, because this method does not rely on the availability of an antibody specific for the therapeutic protein being expressed, it can be easily implemented into any cell line development process.