Structure and Behaviour of Yeast Telomeres

描述（由申请人提供）：长期目标的母基金是了解调控端粒酶在面包酵母。我们希望将这项资助的最初目的扩展到第二个模型系统中的端粒酶分析，即裂殖酵母S。粟球。因此，我们根据NOT-OD-09-058申请资金，通知标题为：NIH宣布恢复法案资金可用于竞争性修订申请。在父母基金支持的工作中，我的实验室率先使用染色质免疫沉淀来研究S。酿酒酵母端粒酶在这里，我们建议使用染色质免疫沉淀来确定是否S。粟酒裂殖酵母端粒酶受细胞周期和端粒长度的调节，如在S.啤酒。我们还将使用三杂交筛选和质谱分析来鉴定S。粟酒端粒酶全酶。我们还提出了一种微观/细胞生物学方法，该方法依赖于双分子荧光互补来研究保护S末端的多蛋白shelterin复合物的相互作用和调节。粟酒裂殖酵母染色体从降解和调节其访问端粒酶。我们希望将我们的端粒研究扩展到S。pombe，因为这种生物体中端粒维持的多个方面比S.啤酒。例如，最近发现的S.粟酒裂殖酵母shelterin复合体与人类端粒处的复合体惊人地相似。最后，还没有系统的遗传筛查S。粟酒裂殖酵母端粒酶组分，因此可能它的一些亚基尚未被鉴定。 公共卫生相关性：端粒酶的调节与人类健康高度相关。端粒酶的表达在绝大多数人类肿瘤中上调，与其无限的生长潜力有关。此外，端粒酶在人类干细胞中以非常低的水平表达，即使该水平的适度降低也会导致干细胞衰竭，对寿命产生可怕的影响。

DESCRIPTION (provided by applicant): The long term goal of the parent grant is to understand the regulation of telomerase in baker's yeast. We wish to extend the original aims of this grant to the analysis of telomerase in a second model system, the fission yeast, S. pombe. Thus, we are requesting funds under NOT-OD-09-058 with Notice Title: NIH announces the availability of recovery act funds for competitive revision applications. In work supported by the parent grant, my lab has pioneered the use of chromatin immuno-precipitation to study S. cerevisiae telomerase. Here we propose to use chromatin immuno-precipitation to determine if S. pombe telomerase is regulated by both the cell cycle and by telomere length, as it is in S. cerevisiae. We will also use both a three-hybrid screen and mass spectrometry to identify new components of the S. pombe telomerase holoenzyme. We also propose a microscopic/cell biological approach that relies on bimolecular fluorescent complementation to study the interaction and regulation of the multi-protein shelterin complex that protects the ends of S. pombe chromosomes from degradation and regulates their access to telomerase. We wish to expand our telomere research to S. pombe because multiple aspects of telomere maintenance in this organism are more similar to humans than is true for S. cerevisiae. For example, the recently discovered S. pombe shelterin complex is strikingly similar to the complex at human telomeres. Finally, there has been no systematic genetic screen for S. pombe telomerase components so it is possible that some of its subunits have not been identified. PUBLIC HEALTH RELEVANCE: The regulation of telomerase is highly relevant to human health. Telomerase expression, which is upregulated in the vast majority of human tumors, is associated with their unlimited growth potential. In addition, telomerase is expressed but at very low levels in human stem cells, and even modest reductions in this level can lead to stem cell failure with dire effects on life span.