Differential Plasmid Representation

Case Western Reserve University, Cleveland OH

Investigators

Abstract

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.