Drosophila Genes Affecting Chromosome Segregation

描述（由申请人提供）：减数分裂或有丝分裂期间染色体分离错误可导致自然流产或异常，如唐氏综合征。它们也与导致癌症和衰老的过程有关。我们的实验室已经进行了大规模的遗传筛选，使用生物体果蝇，以确定基因编码的蛋白质，确保适当的染色体分离的关键。在第一个具体的目标，我们将调查几个新的组件的染色体动粒，首先发现在我们的屏幕和已保守的后生动物，包括人类。该目标的一个焦点领域是包括蛋白质ZW10、Rod和Zwilch的多亚基复合物。该复合物将分子马达动力蛋白靶向于动粒，并且以目前未知的方式需要用于调控后期发作的纺锤体组装检查点的操作。我们提出的实验解剖这个复杂的结构，并调查其与NudE，动力蛋白相关蛋白，其作用目前还不清楚的相互作用。在这一目标的第二个重点领域将探讨的可能性，在我们的遗传筛选中发现的两个新的蛋白质可能共同构成一个新的亚组在着丝粒也需要适当的染色体分离。 第二个具体目标描述了我们实验室的一个新的研究领域。在我们的筛选中发现的果蝇基因之一长城基因的突变，会导致染色体浓缩不足，因为通过前期的延迟。长城基因编码一种新的蛋白激酶，其结构不寻常，在昆虫和脊椎动物中是保守的。我们制备了抗非洲爪蟾长城蛋白的抗体，并将其用于。从卵母细胞提取物中去除这种激酶。令人惊讶的是，Greatwall耗尽阻止了提取物进入或维持M相。在第二个具体目标中，我们将跟踪这些有趣的观察结果，以了解长城在有丝分裂进程中的作用。我们提出的实验，以确定长城的基板以及上游激酶参与长城激活有丝分裂过程中。其他实验将调查长城的卵母细胞成熟的可能的发展作用，并将寻找其他蛋白质与这种激酶协会。对长城的拟议研究有可能提供关于脊椎动物细胞有丝分裂进入的独特机制见解。

DESCRIPTION (provided by applicant): 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 processes leading to cancer and aging. Our laboratory has undertaken large-scale genetic screens using the organism Drosophila melanogaster to identify genes encoding proteins critical for ensuring proper chromosome segregation. In the first specific aim, we will investigate several novel components of the chromosomal kinetochores that were first found in our screens and that have been conserved among metazoans including humans. One area of focus in this aim is a multisubunit complex including the proteins ZW10, Rod, and Zwilch. This complex targets the molecular motor dynein to the kinetochore, and it is required in a currently unknown fashion for the operation of the spindle assembly checkpoint that regulates anaphase onset. We propose experiments to dissect the structure of this complex and to investigate its interactions with NudE, a dynein-associated protein whose role is currently not well understood. A second area of focus in this aim will explore the possibility that two new proteins identified in our genetic screens may together constitute a new subassembly at the kinetochore also needed for proper chromosome segregation. The second specific aim describes a new area of research for our laboratory. Mutations in greatwall, one of the Drosophila genes found in our screens, cause chromosome undercondensation because of delayed transit through prophase. The greatwall gene encodes a novel protein kinase with an unusual structure that is conserved in insects and vertebrates. We made antibody against the Xenopus Greatwall protein and used it to. deplete this kinase from oocyte extracts. To our surprise, Greatwall depletion prevented the extracts from entering or maintaining M phase. In the second specific aim, we will follow up these intriguing observations in order to understand Greatwall's role in mitotic progression. We propose experiments to define Greatwall's substrates as well as the upstream kinases involved in Greatwall activation during mitosis. Additional experiments will investigate Greatwall's possible developmental role in oocyte maturation and will search for other proteins with which this kinase associates. The proposed studies on Greatwall have the potential to offer unique mechanistic insights concerning mitotic entry in vertebrate cells.