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唐氏综合症等异常。它们还与导致癌症和衰老的过程有关。我们的实验室使用果蝇进行了大规模的基因筛选，以确定编码蛋白质的基因，这些蛋白质对确保适当的染色体分离至关重要。在第一个特定目标中，我们将研究在我们的筛选中首次发现并在包括人类在内的后生动物中保存的染色体着丝点的几个新组分。该目标的一个重点领域是多亚基复合物，包括蛋白质ZW10， Rod和Zwilch。这种复合物将分子马达动力蛋白靶向着丝点，并且它是调控后期开始的纺锤体组装检查点的操作所必需的，目前尚不清楚。我们建议通过实验来剖析该复合物的结构，并研究其与NudE的相互作用，NudE是一种动力蛋白相关蛋白，其作用目前尚不清楚。本目标的第二个重点领域将探索在我们的遗传筛选中发现的两种新蛋白质可能共同构成着丝点上的新亚组装的可能性，这也是染色体正确分离所需要的。

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.