Structure and Behaviour of Yeast Telomeres

描述（由申请人提供）：父母资助的长期目标是了解面包酵母中端粒酶的调节。我们希望将这项拨款的最初目的扩展到分析第二个模型系统中的端粒酶，裂变酵母，S. pombe。因此，我们在no - od -09-058下申请资金，并附有通知标题：NIH宣布为竞争性修订申请提供恢复法案资金。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的端粒酶。在这里，我们建议使用染色质免疫沉淀来确定S. pombe端粒酶是否受到细胞周期和端粒长度的调节，就像S. cerevisiae一样。我们还将使用三杂交筛选和质谱法来鉴定S. pombe端粒酶全酶的新成分。我们还提出了一种依靠双分子荧光互补的微观/细胞生物学方法来研究多蛋白庇护蛋白复合物的相互作用和调控，该复合物保护S. pombe染色体末端免受降解并调节其端粒酶的获取。我们希望将我们的端粒研究扩展到S. pombe，因为这种生物的端粒维护的多个方面比S. cerevisiae更类似于人类。例如，最近发现的S. pombe庇护蛋白复合物与人类端粒的复合物惊人地相似。最后，对于pombe端粒酶的成分，没有系统的遗传筛选，所以它的一些亚基可能还没有被确定。

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.