EADS, B.D.*; HAND, S.C.: Modulating Mitochondrial Transcription in Artemia franciscana Embryos
Gastrula stage embryos of the brine shrimp Artemia franciscana exhibit developmental and metabolic arrest in response to adverse environmental conditions such as anoxia. This arrest is coordinated and profound, indicating a high degree of control at the level of gene expression. Previous studies have shown an arrest during anoxia of protein synthesis and degradation, with concomitant extension of protein half-life, as well as decreased nuclear transcription. Mitochondria isolated from these embryos provide an ideal system in which to study mechanisms of transcriptional modulation during physiological challenge, including lack of O2 and the lowered ATP concentrations and pHi associated with anoxia. For example, mitochondrial half-life can be measured in organello in a way impossible with isolated nuclei. Using dot blots of mtRNA, mitochondria exposed to anoxia in vitro had significantly longer RNA half-lives than did aerobic mitochondria, indicating degradation is arrested by lack of oxygen. In addition, when exposed to anoxic pHi in vitro, an even greater proportion of the mtRNA was present after 6 h than in anoxic samples, demonstrating that the pH decline during anoxia is more important for halting degradation than is O2 lack per se. We tested the hypothesis that lack of ATP is a mechanism for halting RNA degradation under anoxia by adding 1 mM ATP in vitro to anoxic mitochondria and found no increase, suggesting that lack of ATP is unlikely to be a causal mechanism. We also developed a nuclease protection assay for new transcription initiation events to test whether initiation by isolated mitochondria contributes to overall transcript levels. Preliminary data indicate that this is the case, and that anoxia decreases the amount of de novo initiation.