PROTEIN DEGRADATION DURING MITOSIS AND DEVELOPMENT

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The Principal Investigator for this award will be Dr. Thomas John McGarry. Dr. McGarry received his M.D. and Ph.D. (Genetics) from the University of Chicago. He finished an internal medicine residency at the Massachusetts General Hospital, and a clinical cardiology fellowship at the University of California, San Francisco. He plans to spend most of his time doing research and is trying to establish an independent program studying cell division and differentiation. The intent of the work described here is to identify new proteins that are degraded by the ubiquitin system during Xenopus development. Previous work predicts that such proteins will be important regulatory molecules governing both mitosis and cell determination. For example, the cell cycle is controlled by the degradation of cyclins and cyclin inhibitors. The first goal will be to isolate cDNA clones for proteins that are degraded during mitosis, since mitotic cell extracts that degrade cyclin are readily available. A plasmid cDNA library made from early embryos will be split into pools containing about 100 independent clones. These pools will be transcribed and translated in vitro. The radioactively labelled proteins will be mixed with mitotic and interphase cell extracts. The proteins will then be separated by gel electrophoresis and analyzed by autoradiography. Bands that are stable in interphase extracts but disappear after incubation in mitotic extracts will be identified. Sib-selection protocols will be used to isolate individual cDNA clones that encode degraded proteins from the pools. We will confirm that the proteins encoded by these candidate cDNAs are degraded by the ubiquitin system. Bona fide ubiquitin substrates should be rapidly degraded in an ATP and ubiquitin dependent fashion. The next goal will be to determine the specific function of the proteins which are identified. We expect to find new CDK inhibitors, novel components of the spindle apparatus, and cyclins. We will examine their function in detail using standard biochemical and cytological assays of cell cycle progression. One approach will be to add excess protein to the cell extracts and see how progression is affected. We also hope to construct nondegradable but functional mutants of the proteins, since these would be expected to have dominant effects. The experiments will be extended to isolate clones for proteins that are degraded at specific times in development. For example, proteins degraded during meiosis or gastrulation could be identified by a similar approach, using a cDNA library and cell extracts from oocytes or appropriately staged embryos. These experiments will be done in Marc Kirschner's laboratory at Harvard Medical School. Dr. Kirschner has studied cell division and development in Xenopus embryos for many years and has an impressive publication record. He has a long history of training independent and productive scientists.