DROSOPHILA Genes Affecting Chromosome Segregation

描述(申请人摘要)：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畸形，如 唐氏综合症，它们也与遗传进展有关 导致癌症和衰老。我们的实验室研究了果蝇的几个基因 这是正确的染色体分离所必需的。我们相信这一点 生物在遗传学、基因组学和细胞学方面具有重要的优势 为研究染色体行为和细胞周期提供独特的机会 细胞分裂过程中的进程。 我们首先关注了一种进化上保守的基因，称为zwlO。基因突变 ZwlO不仅扰乱染色体分离，而且还防止 调节后期启动的主轴组件检查点的操作。这个 ZW10蛋白表现出一种不寻常的动态定位模式。 细胞周期，在染色体着丝点和着丝点之间移动 微管。我们发现这种模式受到两极张力的影响 在单个染色体上。我们还发现，ZW10是一个大型 蛋白质复合体，其活性之一是靶向分子马达 动毛虫的动力系统。 第一个具体目标描述了对ZW10的进一步调查。我们将使用 用实时视频显微镜研究ZW10-GFP的分布 细胞周期中的融合蛋白。我们将确定ZW1O的哪些域 它在细胞内分布的各个方面都是必需的 功能。我们将分析双突变组合以将ZW10定位在 纺锤体检查点和其他方面的生化途径 后期发病。我们还将测试zw10突变体的所有方面 表型，包括它对纺锤体检查点的影响，是由 动粒中没有动力蛋白。 第二个具体目标是从生物化学和遗传学的角度描述 大型综合体，其中ZW10是其中的一部分。净化环境污染的初步努力 亲和层析法制备的络合物具有很好的应用前景。我们将使用几个 验证候选蛋白质与ZW10关联的技术。我们会 然后用质谱仪鉴定这些蛋白质，并将确定是否 它们受到翻译后修改的影响。我们的最终目标是 获得针对这些蛋白质的抗体，以及基因突变 对它们进行编码。 第三个具体目标将利用我们在之前的资金中的认同感 一组导致早熟姐妹染色单体的新突变的周期 果蝇的分离、非整倍体或中期停滞。我们会研究 与这些突变相关的表型更详细，选择克隆 突变基因，然后研究细胞内的位置 相应的基因产物。因为我们关注的是基因而不是以前的基因 已知在有丝分裂的任何方面起作用，我们相信基因和抗体 通过拟议的研究获得的试剂将提供一种广泛和独特的 查看后期开始时发生的事件。

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.