Drosophila Genes Affecting Chromosome Segregation

描述(由申请人提供)：减数分裂或有丝分裂过程中染色体分离错误可能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。我们的实验室利用黑腹果蝇进行了大规模的遗传筛查，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。在第一个具体目标中，我们将研究染色体动点的几个新成分，这些成分首先在我们的屏幕上发现，并在包括人类在内的后生动物中保守。这个目标的一个焦点领域是一个多亚单位复合体，包括蛋白质ZW10、Rod和Zwilch。这种复合体将分子马达dynein靶向动粒，并且它是调节后期开始的纺锤体组装检查点操作所需的一种目前未知的方式。我们建议进行实验来剖析这个复合体的结构，并研究它与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。这一目标的第二个重点领域将探索在我们的基因筛查中发现的两种新蛋白质可能共同构成着染色体正常分离所需的着丝粒上的新亚组。 第二个具体目标描述了我们实验室的一个新的研究领域。长城基因是在我们的筛查中发现的果蝇基因之一，它的突变导致染色体凝集不足，因为延迟了通过前期的运输。长城基因编码一种新的蛋白激酶，具有一种在昆虫和脊椎动物中保守的不同寻常的结构。我们制作了针对非洲爪哇长城蛋白的抗体，并将其用于。从卵母细胞提取液中耗尽这种激酶。令我们惊讶的是，长城的枯竭阻止了提取物进入或维持M期。在第二个具体目标中，我们将跟踪这些有趣的观察，以了解长城在有丝分裂进程中的作用。我们建议通过实验来确定长城的底物以及在有丝分裂过程中参与长城激活的上游激酶。更多的实验将研究长城在卵母细胞成熟中可能的发育作用，并将寻找与该激酶相关的其他蛋白质。关于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.