Structure and Behaviour of Yeast Telomeres

描述(申请人提供)：家长资助的长期目标是了解面包酵母中端粒酶的调节。我们希望将这项资助的最初目标扩展到第二个模型系统--裂解酵母S.pombe中的端粒酶分析。因此，我们在NOT-OD-09-058项下请求资金，通知标题：NIH宣布恢复法案资金可用于竞争性修订申请。在父母资助的工作中，我的实验室率先使用染色质免疫沉淀来研究酿酒酵母端粒酶。在这里，我们建议使用染色质免疫沉淀来确定是否像酿酒酵母一样，端粒酶受细胞周期和端粒长度的调节。我们还将使用三杂交筛选和质谱仪来鉴定S.pombe端粒酶全酶的新成分。我们还提出了一种依赖于双分子荧光互补的显微/细胞生物学方法来研究多蛋白保护素复合体的相互作用和调控，该复合体保护S.pombe染色体末端不被降解，并调节它们对端粒酶的访问。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与人类更相似，而不是S.cerevisiae。例如，最近发现的S.pombe Shelterin复合体与人类端粒的复合体惊人地相似。最后，目前还没有对S.pombe端粒酶成分进行系统的遗传筛查，因此它的一些亚单位可能还没有被鉴定出来。 公共卫生相关性：端粒酶的调节与人类健康高度相关。端粒酶在绝大多数人类肿瘤中表达上调，与肿瘤的无限生长潜力有关。此外，端粒酶在人类干细胞中也有表达，但水平非常低，即使这一水平的轻微下降也会导致干细胞衰竭，对寿命造成严重影响。

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.