DROSOPHILA Genes Affecting Chromosome Segregation

描述（申请人的摘要）： 减数分裂或有丝分裂可导致自然流产或异常， 唐氏综合症，他们也被牵连在遗传进展 导致癌症和衰老。我们的实验室研究果蝇的几个基因 是染色体分离所必需的。我们认为这 生物体在遗传学、基因组学和细胞学方面具有重要的优势， 为研究染色体行为和细胞周期提供了独特的机会 在细胞分裂过程中。 我们首先关注一种称为zwlO的进化上保守的基因。突变 zwlO不仅破坏染色体分离，而且它们还阻止染色体的分离。 纺锤体组装检查点的操作，调节后期开始。的 ZW 10蛋白在细胞周期中显示出一种不寻常的动态定位模式。 细胞周期，在染色体动粒和动粒之间移动 微管我们发现这种模式受到两极紧张的影响 在单个染色体上。我们还发现，ZW 1 O是一个大的 蛋白质复合物，其活动之一是靶向分子马达 动力蛋白到动粒。 第一个具体目标描述了对ZW 10的进一步研究。 实时视频显微镜来表征ZW 1 O-GFP的分布 融合蛋白在细胞周期中的作用。我们将确定ZW 1 O的哪些域 是其细胞内分布的各个方面所必需的， 功能我们将分析双突变体组合，以将ZW 10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zw 10突变体的所有方面是否 表型，包括其对纺锤体检查点的影响，是由 动粒处动力蛋白缺失。 第二个具体目标是从生物化学和遗传学角度描述 ZW 1 O是其中的一部分。初步努力净化 通过亲和色谱法制备复合物已经是有希望的。我们将使用几个 技术来验证候选蛋白与ZW 1 O的关联。我们将 然后用质谱法鉴定这些蛋白质， 它们经历翻译后修饰。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 编码它们。 第三个具体目标将利用我们在以前的资金中的识别 一组新的突变导致姐妹染色单体早熟的时期 分离、非整倍体或中期停滞。我们会研究 与这些突变相关的更详细的表型，克隆选择 突变基因，然后研究细胞内的位置， 相应的基因产物。因为我们关注的是基因， 已知在有丝分裂的任何方面起作用，我们认为遗传和抗体 拟议研究获得的试剂将提供更广泛和独特的 在后期开始时发生的事件的观点。

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.