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

用于非分裂酵母细胞的双杂交系统

• 批准号: 6487838
• 负责人: MARGRET C. WERNER-WASHBURNE
• 金额: $ 3万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6487838
• 关键词: DNA replication; beta galactosidase; cell cycle; flow cytometry; gene expression; genetic library; genetic promoter element; genetic transcription; genetic translation; green fluorescent proteins; method development; protein kinase A; protein protein interaction; recombinase; yeast two hybrid system; yeasts

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.

描述（申请人的摘要）：检测蛋白质蛋白质的能力 活细胞中的相互作用一直是deve opment的关键组成部分 基因组分析。酵母双杂交系统已用于检测和 研究许多类型的相互作用。例如在蛋白质，蛋白质和DNA之间， 来自许多不同生物的蛋白质和肽等。这项技术有 以多种方式为人类健康做出贡献的潜力，包括 对细胞功能，结构和信号有更好的了解 转导导致鉴定新型抗生素和 治疗学。当前用于检测相互作用蛋白的双杂交技术 在酵母中，需要在生长细胞中进行相互作用，并且 相互作用足够稳定以提供相对恒定的转录 激活厌倦了生长时期。结果，互动不能是 检测到仅在非分散细胞中发生的，需要特定于逮捕 一个或两个伴侣的修改需要产生的其他蛋白质 仅在非生长细胞中，或者是短暂的。因为大多数细胞花费 他们在非分歧状态的大部分时间，因为新型互动 已知发生这种情况的事件，当检测相互作用的能力 细胞不是分裂对于理解的范围至关重要 活细胞中发生的蛋白质蛋白相互作用。虽然更多 困难，检测瞬态相互作用的能力也很重要， 特别是为了理解诸如信号转导和 代谢。为了解决当前技术的局限性，我们建议 为了开发改良的两杂化测定，将允许检测分子 相互作用，包括仅发生在非分裂细胞中或 相对瞬态。依赖营地的调节和催化亚基 蛋白激酶，分别由BCY1和TPK1-3基因编码 用于测试和完善该系统。选择这些蛋白质是因为BCYLP TPKLP彼此相互作用，并在新型蛋白质复合物中发现 固定相酵母细胞。另外，与BCY IP相互作用的蛋白质 尚未通过经典的两性杂交分析检测到静态细胞。 因为已知TPK催化亚基与数字瞬时相互作用 底物，TPK基因将用于确定是否修改系统 可以适应用于检测瞬态相互作用。一旦建立 酵母，此方法在多种细胞类型中应很有用 促进蛋白质 - 蛋白质相互作用的基因组规模分析 生长的所有阶段。