PROTEIN DEGRADATION DURING MITOSIS AND DEVELOPMENT

有丝分裂和发育过程中的蛋白质降解

• 批准号: 2771162
• 负责人: THOMAS J MCGARRY
• 金额: $ 8.54万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771162
• 关键词: Xenopus; Xenopus oocyte; autoradiography; cell cycle; cell growth regulation; cyclin dependent kinase; cyclins; enzyme inhibitors; genetic library; mitotic spindle apparatus; molecular cloning; protein degradation; ubiquitin; vertebrate embryology

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.

该奖项的主要研究者将是托马斯约翰博士 麦加里 McGarry博士获得了医学博士学位。和博士（遗传学）从 芝加哥大学。 他完成了一个内科住院医师在 马萨诸塞州总医院，并在 加州大学旧金山分校弗朗西斯科。 他计划花大部分时间 他的时间做研究，并试图建立一个独立的计划， 研究细胞分裂和分化。 这里描述的工作的目的是鉴定新的蛋白质， 在非洲爪蟾发育过程中被泛素系统降解。 以前的工作预测，这些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。 比如说， 细胞周期由细胞周期蛋白和细胞周期蛋白的降解控制 抑制剂的 第一个目标将是分离蛋白质的cDNA克隆， 在有丝分裂过程中降解，因为降解细胞周期蛋白的有丝分裂细胞提取物 都是现成的。 利用早期胚胎构建质粒cDNA文库 将被分成包含大约100个独立克隆的池。 这些 库将在体外转录和翻译。 放射性 标记的蛋白质将与有丝分裂和间期细胞混合 萃取物 然后通过凝胶电泳分离蛋白质， 通过放射自显影术分析。在相间提取物中稳定的条带 但在有丝分裂提取物中孵育后消失。 将使用同胞选择方案分离单个cDNA克隆 编码从水池中降解的蛋白质。 我们将确认， 由这些候选cDNA编码的蛋白质被泛素降解 系统真正的泛素底物应该在一个 ATP和泛素依赖方式。 下一个目标将是确定蛋白质的具体功能 它们被识别。 我们期望找到新的CDK抑制剂， 主轴装置的部件和循环。 我们将检查他们的 使用标准的生物化学和细胞学分析， 细胞周期进程 一种方法是将过量的蛋白质添加到 细胞提取物，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功能性突变体，因为 预计这些将产生主要影响。 实验将扩展到分离蛋白质的克隆， 在发育的特定时期退化。 例如蛋白质 在减数分裂或原肠胚形成过程中降解的蛋白质可以通过类似的 方法，使用cDNA文库和来自卵母细胞的细胞提取物， 合适的胚胎阶段。 这些实验将在哈佛的马克·科施纳实验室进行 医学院 Kirschner博士研究了细胞分裂和发育 在非洲爪蟾胚胎中研究多年， 记录 他有着悠久的独立和富有成效的训练历史 科学家