A TWO-HYBRID SYSTEM FOR USE IN NON-DIVIDING YEAST CELLS

描述(申请人摘要)：检测蛋白质-蛋白质的能力 活细胞中的相互作用一直是这一发展的关键组成部分 基因组分析的结果。酵母菌双杂交系统已用于检测和鉴定 研究多种类型的交互作用。例如在蛋白质、蛋白质和DNA之间， 来自许多不同生物体的蛋白质和多肽等。这项技术已经 以多种方式促进人类健康的潜力，包括 提供对细胞功能、结构和信号的更好理解 转导导致鉴定新的抗生素和 治疗学。目前检测相互作用蛋白的双杂交技术 在酵母中需要在生长的细胞中发生相互作用，并且 相互作用足够稳定，以提供相对恒定的转录 在生长期内被激活。因此，交互不能 检测到仅发生在未分裂细胞中，需要特定于逮捕 一个或两个伴侣的修饰，需要产生其他蛋白质 只在不生长的细胞中存在，或者是暂时的。因为大多数细胞花在 它们的大部分时间都处于不分状态，因为新的相互作用 已知发生在这些时间段，检测相互作用的能力 单元格不分割对于理解 发生在活细胞中的蛋白质-蛋白质相互作用。虽然更多 尽管难度很大，但检测瞬时相互作用的能力也很关键， 尤其是对于理解信号转导和信号传导等过程 新陈代谢。为了解决当前技术的局限性，我们建议 开发一种改进的双杂交试验，将允许检测分子 相互作用，包括仅在未分裂的细胞中发生的或 相对短暂的。CAMP依赖的调节和催化亚基 分别由BCY1和TPK1-3基因编码的蛋白激酶将被激活 用于测试和改进此系统。之所以选择这些蛋白质是因为Bcylp 和TpkLp相互作用，并在新的蛋白质复合体中发现 固定相酵母细胞。此外，与Bcy Ip相互作用的蛋白质在 经典的双杂交分析没有检测到静止细胞。 因为已知Tpk催化亚基与数个 在底物中，将使用TPK基因来确定修改后的系统是否 可适用于检测瞬时交互。一旦确定是为了 酵母，这种方法应该在广泛的细胞类型中有用，以 促进蛋白质-蛋白质相互作用的基因组尺度分析 所有的成长阶段。

DESCRIPTION (Applicant's abstract): The ability to detect protein-protein interactions in living cells has been a critical component in the deve]opment of genomic analyses. The yeast two-hybrid system has been used to detect and study many types of interactions. e.g. between proteins, proteins and DNA, proteins and peptides etc. from many different organisms. This technology has the potential to contribute to human health in numerous ways, including providing a better understanding of cellular function, structure, and signal transduction leading to the identification of novel antibiotics and theraputics. Current two-hybrid technologies for detecting interacting proteins in yeast require that interactions take place in growing cells and that the interactions be stable enough to provide relatively constant transcriptional activation dunng the period of growth. As a result, interactions cannot be detected that occur only in non-dividing cells, require arrest-specific modifications of one or both partners, require other proteins that are produced only in non-growing cells, or are transient. Because most cells spend the majority of their time in non-dividing states and because novel interactions are known to occur dunng these times, the ability to detect interactions when cells are not dividing is critical for understanding the range of protein-protein interactions that occur in living cells. Although more difficult, the ability to detect transient interactions is also critical, especially for understanding processes such as signal transduction and metabolism. To address the limitations of the current technologies, we propose to develop a modified two-hybrid assay that will allow detection of molecular interactions, including those that occur only in non-dividing cells or are relatively transient. The regulatory and catalytic subunits of cAMP-dependent protein kinase, encoded by the BCY1 and TPK1-3 genes, respectively, will he used to test and refine this system. These proteins were selected because Bcylp and Tpklp interact with each other and are found in novel protein complexes in stationary-phase yeast cells. Also, the proteins that interact with Bcy Ip in quiescent cells have not been detected by classical two-hybrid analysis. Because Tpk catalytic subunits are known to interact transiently with a number of substrates, TPK genes will be used to determine whether the modified system can be adapted for detecting transient interactions. Once established for yeast, this method should be useful in a broad range of cell types to facilitate the genomic-scale analysis of protein-protein interactions during all phases of growth.