DROSOPHILA Genes Affecting Chromosome Segregation

影响染色体分离的果蝇基因

• 批准号: 6710143
• 负责人: MICHAEL L GOLDBERG
• 金额: $ 37.32万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6710143
• 关键词: Drosophilidae; affinity chromatography; cell cycle; chromosome movement; cytogenetics; gene expression; gene interaction; gene mutation; genetic crossing over; immunofluorescence technique; laboratory rabbit; mass spectrometry; matrix assisted laser desorption ionization; molecular cloning; posttranslational modifications; protein structure function; protein transport; transcription factor; video microscopy

DESCRIPTION (Applicant's Abstract): Mistakes in chromosome segregation during meiosis or mitosis can lead to spontaneous abortion or to abnormalities such as Down syndrome, They have also been implicated in the genetic progressions leading to cancer and aging. Our laboratory studies several genes in Drosophila that are required for proper chromosome segregation. We believe that this organism offers important advantages in genetics, genomics and cytology that provide unique opportunities to investigate chromosome behavior and cell cycle progression during cell division. We first focused on an evolutionarily conserved gene called zwlO. Mutations in zwlO not only disrupt chromosome segregation, but they also prevent the operation of the spindle assembly checkpoint that regulates anaphase onset. The ZW1O protein displays an unusual, dynamic pattern of localization during the cell cycle, moving between chromosomal kinetochores and kinetochore microtubules. We found that this pattern is influenced by bipolar tension across individual chromosomes. We also discovered that ZW1O is part of a large protein complex, one of whose activities is to target the molecular motor dynein to the kinetochore. The first specific aim describes further investigations on ZW1O.We will use real-time video microscopy to characterize the distribution of a ZW1O-GFP fusion protein during the cell cycle. We will determine which domains of ZW1O are required for various aspects of its intracellular distribution and function. We will analyze double mutant combinations to position ZW1O in the biochemical pathways underlying the spindle checkpoint and other aspects of anaphase onset. We will also test whether all aspects of the zw10 mutant phenotype, including its effects on the spindle checkpoint, are caused by the absence of dynein at the kinetochore. The second specific aim is to characterize biochemically and genetically the large complex of which ZW1O is a part. Preliminary efforts to purify the complex by affinity chromatography have been promising. We will use several techniques to verify the association of candidate proteins with ZW1O. We will then identify these proteins by mass spectrometry, and will determine whether they are subject to post-translational modification. Our ultimate goal is to obtain antibodies against these proteins, as well as mutations in the genes encoding them. The third specific aim will exploit our identification in the previous funding period of a set of new mutations that cause precocious sister chromatid separation, aneuploidy, or metaphase arrest in Drosophila. We will study the phenotypes associated with these mutations in more detail, clone selected mutant genes, and then investigate the intracellular location of the corresponding gene products. Because we are focusing on genes not previously known to function in any aspect of mitosis, we believe the genetic and antibody reagents obtained by the proposed studies will provide a broadened and unique view of the events occurring at anaphase onset.

描述（申请人的摘要）：染色体分离过程中的错误 减数分裂或有丝分裂可导致自然流产或异常，例如 唐氏综合症，它们也与遗传进展有关 导致癌症和衰老。我们的实验室研究果蝇的几个基因 这是正确染色体分离所必需的。我们相信，这 生物体在遗传学、基因组学和细胞学方面具有重要的优势 提供独特的机会来研究染色体行为和细胞周期 细胞分裂过程中的进展。 我们首先关注一个名为 zwlO 的进化保守基因。突变在 zwlO 不仅破坏染色体分离，而且还阻止 调节后期开始的主轴组件检查点的操作。这 ZW1O 蛋白在 细胞周期，在染色体着丝粒和着丝粒之间移动 微管。我们发现这种模式受到双向紧张的影响 跨个体染色体。我们还发现 ZW1O 是一个大型 蛋白质复合物，其活性之一是靶向分子马达 动力蛋白至着丝粒。 第一个具体目标描述了对 ZW1O 的进一步调查。我们将使用 实时视频显微镜来表征 ZW1O-GFP 的分布 细胞周期中的融合蛋白。我们将确定 ZW1O 的哪些域 其细胞内分布的各个方面都需要 功能。我们将分析双突变体组合以将 ZW1O 定位在 纺锤体检查点和其他方面的生化途径 后期开始。我们还将测试zw10突变体的各个方面是否 表型，包括其对纺锤体检查点的影响，是由 着丝粒处缺乏动力蛋白。 第二个具体目标是从生化和遗传学角度表征 ZW1O 是其中的一部分的大型综合体。初步净化的努力 通过亲和层析法制备复合物已很有前景。我们将使用几个 验证候选蛋白与 ZW1O 关联的技术。我们将 然后通过质谱鉴定这些蛋白质，并确定是否 它们会受到翻译后修饰。我们的最终目标是 获得针对这些蛋白质以及基因突变的抗体 对它们进行编码。 第三个具体目标将利用我们在先前融资中的识别 导致姐妹染色单体早熟的一组新突变的时期 果蝇的分离、非整倍体或中期停滞。我们将研究 与这些突变相关的更详细的表型，克隆选择 突变基因，然后研究其在细胞内的位置 相应的基因产物。因为我们现在关注的不是以前的基因 已知在有丝分裂的任何方面发挥作用，我们相信遗传和抗体 通过拟议的研究获得的试剂将提供更广泛和独特的 对后期开始时发生的事件的看法。