Differential Plasmid Representation

许多表型上的“沉默”突变（例如基因X的缺失，或突变蛋白的存在）与存活是相容的，因为细胞合成适当的补偿蛋白并避免表达其他蛋白。通过识别这些功能相关的蛋白质，人们可以表征Xp的生物学意义，从而开发出间接调节其参与的功能的方法。该项目将开发一种广泛适用的方法来鉴定这些蛋白质，使用“差分质粒表征”（DPR）。DPR将首先用于比较野生型酵母和生长良好但不合成跨膜蛋白Wsc1p的等基因突变体。经典的方法已经描述了Wsc1p，它被认为是一个“压力传感器”，并启动信号传导到“细胞完整性”途径。wt和突变株都将用着丝粒cDNA文库进行转化。然后，在诱导每个细胞表达单个异位cDNA的条件下，在选择性培养基中培养转化体的混合物。这将引起许多转化者之间的竞争，这些转化者构成了一套不同的遗传背景。在10-20代之后，将从两种类型的转化体中回收DNA，以便使用DNA微阵列确定最初存在的每种质粒的相对丰度。如果每个质粒没有提供生长优势或劣势，则应保持其初始相对丰度。因此，比较wt和突变菌株的富集/耗尽数据将强有力地预测哪些cdna与Wsc1p的缺失表现出正或负的“合成”关系。这些具有重要功能的cdna预计将与那些已知与细胞完整性途径相关的dna重叠——至少部分重叠。本项目将为开发这种新方法提供初步数据。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比鉴定基因间“合成致死”相互作用的经典方法（在这种方法中，如果第一个基因突变或删除，第二个基因必须继续转录）要简单和快速得多。它还应该使检测一类遗传相互作用成为可能，这类遗传相互作用是用经典方法难以获得的。在这些“合成生存”的相互作用中，只有在另一个基因也发生突变的情况下，一个基因的突变才能与生存相容。后续的研究将使用DPR来研究其他酵母基因缺失或特定突变蛋白表达的后果。这种策略的适应性应该适用于研究许多可以被敲除而没有明显效果的小鼠基因。基于相同方法的实验也应该有助于组织工程和细胞对环境和遗传压力的反应的研究。这些新颖的实验策略的发展将立即对高中生、大学生、博士生以及研究者实验室的博士后具有教育意义。随着这些新方法被广泛接受，其教育和实用价值将得到极大的扩展。这个项目的目的不是解决一个单一的生物学难题，而是旨在开发一种广泛适用的方法。

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.