PROTEIN DEGRADATION DURING MITOSIS AND DEVELOPMENT

该奖项的首席研究员将是托马斯·约翰博士 麦克格里。McGarry博士获得医学博士和博士学位(遗传学) 芝加哥大学。他在西雅图大学完成了内科实习 马萨诸塞州总医院和临床心脏病学研究员 加州大学旧金山分校。他计划花大部分时间 他花时间做研究，正试图建立一个独立的项目 研究细胞分裂和分化。 这里描述的工作的目的是识别新的蛋白质， 在非洲爪哇发育过程中被泛素系统降解。 先前的工作预测，这种蛋白质将是重要的调控蛋白。 控制有丝分裂和细胞决定的分子。例如, 细胞周期受细胞周期蛋白和细胞周期蛋白的降解控制 抑制剂。 第一个目标是分离出蛋白质的cdna克隆。 在有丝分裂过程中降解，因为有丝分裂细胞提取物能降解周期蛋白 都是现成的。以早期胚胎为材料构建的质粒库 将被分成包含大约100个独立克隆的池。这些 POOL将在体外转录和翻译。有放射性的 标记的蛋白质将与有丝分裂细胞和间期细胞混合 萃取物。然后用凝胶电泳法分离蛋白质和 通过放射自显影进行分析。在相间萃取物中稳定的条带 但在有丝分裂提取物中孵育后消失将被鉴定。 将使用SIB选择协议来分离单个cDNA克隆 对池中降解的蛋白质进行编码。我们将确认 这些候选cDNA编码的蛋白质被泛素降解 系统。真正的泛素底物应该在 ATP和泛素依赖的方式。 下一个目标将是确定蛋白质的特定功能 它们被识别出来了。我们希望找到新的CDK抑制剂，新的 主轴装置的组件，以及周期蛋白。我们将审查他们的 使用标准的生化和细胞学分析的详细功能 细胞周期进程。一种方法是将多余的蛋白质添加到 细胞提取出来，看看进展如何受到影响。我们也希望 构建蛋白质的不可降解但有功能的突变体，因为 预计这些措施将产生显性影响。 这些实验将扩大到分离蛋白质的克隆 在发育过程中的特定时间退化。例如，蛋白质 在减数分裂或原肠分裂过程中降解的可以通过类似的 方法，使用cdna文库和从卵母细胞或 适当分期的胚胎。 这些实验将在哈佛大学马克·基什内尔的实验室里进行 医学院。Kirschner博士研究了细胞分裂和发育 在非洲爪哇胚胎研究多年，并有一本令人印象深刻的出版物 唱片。他在训练独立和富有成效方面有很长的历史 科学家。

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.