No abstract

Using a synthetic lethality screen we found that the Sit4 phosphatase is functionally linked to the ubiquitin-proteasome system. Yeast cells harboring sit4 mutations and an impaired proteasome (due to pre1-1 pre4-1 mutations) exhibited defective growth on minimal medium. Nearly identical synthetic effects were found when sit4 mutations were combined with defects of the Rad6/Ubc2- and Cdc34/Ubc3-dependent ubiquitination pathways. Under synthetic lethal conditions, sit4 pre or sit4 ubc mutants formed strongly enlarged unbudded cells with a DNA content of 1N, indicating a defect in the maintenance of cell integrity during starvation-induced G1 arrest. Sit4-related synthetic effects could be cured by high osmotic pressure or by the addition of certain amino acids to the growth medium. These results suggest a concerted function of the Sit4 phosphatase and the ubiquitin-proteasome system in osmoregulation and in the sensing of nutrients. Further analysis showed that Sit4 is not a target of proteasome-dependent protein degradation. We could also show that Sit4 does not contribute to regulation of proteasome activity. These data suggest that both Sit4 phosphatase and the proteasome act on a common target protein.

We present a new free-flow electrophoretic separation system that extends the established concepts of nucleic acid migration in an electric field to a broadly applicable preparative scale. The system comprises a disposable flow tube in which the target nucleic acids are separated from impurities by a balanced combination of electrophoretic migration and counter-streaming electroosmotic flow under the influence of an applied external electric field. Despite the complex theoretical background the introduced electrophoretic technology offers simple hardware setup and handling protocols. A variable number of small and disposable flow tubes can be processed in parallel, which largely eliminates the cumulative increase in extraction times inherent to batch processing methods and allows faster throughput of intermediate sample numbers. We demonstrate easy isolation of nucleic acids without user interaction during the run by using existing and well established lysis chemistries. Sample loading is realized by concentrated transfer of DNA-loaded magnetic beads from a lysis reaction into the extraction flow tube. The present study centers on the development of a functional model for the device and the flow tube as well as a preliminary standard extraction protocol. The system is compatible with a broad range of sample types and we present proof of principle data demonstrating its suitability for biomarker detection in translational research applications.