Differential Plasmid Representation

差异质粒表达

• 批准号: 0431496
• 负责人: Alan Tartakoff
• 金额: --
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0431496&HistoricalAwards=false
• 关键词: Differential; Plasmid; Representation

Many phenotypically "silent" mutations (e.g. deletion of gene X, or the presence of a mutant protein) are compatible with survival since cells synthesize appropriate compensatory proteins and avoid the expression of others. By identifying these functionally related proteins, one can characterize the biological significance of Xp, and therefore develop indirect means of regulating functions in which it participates. This project will develop a broadly-applicable method for identification of such proteins, using "Differential Plasmid Representation" (DPR). DPR will first be used to compare wild type yeast to an isogenic mutant which grows well but does not synthesize the transmembrane protein, Wsc1p. Classical approaches have already characterized Wsc1p, which is thought to function as a "stress sensor" and initiate signaling to the "cell integrity" pathway. Both wt and mutant strains will be transformed with a centromeric cDNA library. The mixture of transformants will then be grown in selective medium under conditions which induce expression of a single ectopic cDNA in each cell. This will cause competition among the many transformants, which constitute a diverse set of genetic backgrounds. After 10-20 generations, the cDNAs will be recovered from both types of transformants in order to determine - using DNA microarrays - the relative abundance of each of the plasmids which was originally present. Each plasmid should retain its initial relative abundance if it provides no growth advantage or disadvantage. Comparison of the enrichment/depletion data for wt vs mutant strains will therefore make strong predictions as to which cDNAs exhibit a positive or negative "synthetic" relation with deletion of Wsc1p. The functionally significant cDNAs are expected to overlap - at least in part - with those which are already known to be related to the cell integrity path.The present project will generate preliminary data to develop this novel method.DPR will allow identification of multiple genes which contribute - to differing degrees - to the ability of cells to tolerate "silent" mutations. DPR is substantially simpler and faster than classical approaches for identification of "synthetic lethal" interactions between genes (in which a second gene must continue to be transcribed if a first gene is mutated or deleted). It should also make it possible to detect a class of genetic interactions which is less accessible by classical methods. In these "synthetic survival" interactions, mutation of one gene is compatible with survival only if a second gene is also mutated.Later studies will use DPR to investigate the consequences of deletion of other yeast genes or the expression of specific mutant proteins. Adaptations of this strategy should be applicable to investigation of the many murine genes which can be knocked out without apparent effect. Experiments based on the same methodologies should also facilitate investigations of tissue engineering and the responses of cells to environmental and genetic stress. Development of these novel experimental strategies will be immediately educational for the group of high school, college, and PhD students, as well as postdoctoral fellows, in the investigator's laboratory. As these novel methods become widely accepted, the educational and practical value will extend considerably. This project does not aim to solve a single biological puzzle, but is instead designed to develop a broadly applicable method.

许多表型“沉默”突变（例如基因 X 的缺失或突变蛋白的存在）与生存相容，因为细胞合成适当的补偿蛋白并避免其他蛋白的表达。 通过鉴定这些功能相关的蛋白质，人们可以表征 Xp 的生物学意义，从而开发出调节其参与的功能的间接方法。 该项目将使用“差异质粒表达”（DPR）开发一种广泛适用的方法来鉴定此类蛋白质。 DPR 将首先用于将野生型酵母与生长良好但不合成跨膜蛋白 Wsc1p 的同基因突变体进行比较。 经典方法已经描述了 Wsc1p 的特征，它被认为具有“压力传感器”的功能，并启动向“细胞完整性”途径发出信号。 野生型和突变型菌株都将用着丝粒 cDNA 文库转化。 然后将转化体的混合物在选择性培养基中在诱导每个细胞中单个异位cDNA表达的条件下生长。 这将导致许多转化体之间的竞争，这些转化体构成了不同的遗传背景。 10-20代后，将从两种类型的转化体中回收cDNA，以便使用DNA微阵列确定最初存在的每种质粒的相对丰度。 如果每个质粒不提供生长优势或劣势，则应保留其初始相对丰度。 因此，野生型与突变株的富集/消除数据的比较将做出关于哪些 cDNA 与 Wsc1p 缺失表现出正或负“合成”关系的强有力的预测。 功能上重要的 cDNA 预计会与已知与细胞完整性路径相关的 cDNA 至少部分重叠。本项目将产生初步数据来开发这种新方法。DPR 将允许识别多个基因，这些基因在不同程度上有助于细胞耐受“沉默”突变的能力。 DPR 比识别基因之间“合成致死”相互作用的经典方法要简单、快捷得多（其中，如果第一个基因突变或删除，第二个基因必须继续转录）。 它还应该使得检测一类通过经典方法难以实现的遗传相互作用成为可能。 在这些“综合生存”相互作用中，只有当第二个基因也发生突变时，一个基因的突变才能与生存兼容。后续研究将使用 DPR 来研究删除其他酵母基因或特定突变蛋白表达的后果。 这种策略的适应应该适用于许多小鼠基因的研究，这些基因可以被敲除而没有明显的影响。 基于相同方法的实验也应有助于组织工程以及细胞对环境和遗传应激的反应的研究。 这些新颖的实验策略的开发将立即对研究者实验室的高中生、大学生和博士生以及博士后研究员具有教育意义。 随着这些新颖的方法被广泛接受，其教育和实用价值将大大扩展。 该项目的目的并不是解决单一的生物学难题，而是旨在开发一种广泛适用的方法。