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发展中的关键组成部分 基因组分析。酵母双杂交系统已被用于检测和 研究多种类型的相互作用。例如蛋白质、蛋白质和DNA之间， 蛋白质和肽等。该技术具有 以多种方式促进人类健康的潜力，包括 更好地了解细胞功能、结构和信号 转导导致新抗生素的鉴定， 治疗学检测相互作用蛋白质的双杂交技术 在酵母中需要在生长细胞中发生相互作用， 相互作用足够稳定以提供相对恒定的转录 在生长期激活。因此，相互作用不能被 仅在非分裂细胞中检测到，需要特定的抑制 一个或两个伴侣的修饰，需要产生其他蛋白质， 仅在非生长细胞中，或者是瞬时的。因为大多数细胞 大部分时间处于非分裂状态， 已知在这些时候会发生，检测相互作用的能力， 细胞不分裂对于理解细胞的范围至关重要。 在活细胞中发生的蛋白质-蛋白质相互作用。尽管更多 困难，检测瞬时相互作用的能力也是至关重要的， 特别是对于理解诸如信号转导和 新陈代谢.为了解决现有技术的局限性，我们建议 开发一种改良的双杂交检测方法， 相互作用，包括那些只发生在非分裂细胞或 相对短暂。cAMP依赖的调节和催化亚基 分别由BCY 1和TPK 1 -3基因编码的蛋白激酶将被激活。 用来测试和完善这个系统选择这些蛋白质是因为Bcylp 和Tpklp相互作用，并发现在新的蛋白质复合物中， 静止期酵母细胞。此外，与Bcy Ip相互作用的蛋白质在 通过经典的双杂交分析还没有检测到静止细胞。 因为已知Tpk催化亚基与许多 底物，TPK基因将被用来确定是否修饰系统 可以适用于检测瞬时相互作用。一旦建立， 酵母，这种方法应该适用于广泛的细胞类型， 促进蛋白质-蛋白质相互作用的基因组规模分析， 生长的各个阶段。

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.