第七章 蛋白质相互作用.pptVIP

* And then the blue highlights here show proteins for which we have interaction data.. I’m not going to describe these ints in the detail that I did last year, but we saw a nice pattern where the assay was confirming several established ints, such as the PI3Ks and several of the ints over on the right involving the actin cytoskeleton.. But also providing novel data such as PLC gamma 2 binding SHIP and also this adapter protein Ruk1 which has been proposed to regulate PI3K activity.. * * * * * 研究阻断大分子间特定相互作用的机理 特点：利用反向筛选报告基因，即蛋白间特异相互作用所激活的报告基因能阻碍宿主菌的生长，从而发现蛋白间结合的解离因素。 All reverse systems make use of yeast strains in which expression of interacting hybrid proteins increases the expression of a counterselectable marker that is toxic under particular conditions. Under these conditions, dissociation of an interaction provides a selective advantage, thereby facilitating detection: a few growing yeast colonies in which hybrids fail to interact can be identified among millions of non-growing colonies expressing interacting proteins. The first reverse two-hybrid system uses a yeast strain that is resistant to cycloheximide due to the presence of a mutant CYH2 gene. This strain also contains the wild-type CYH2 allele under the transcriptional control of the GAL1 promoter. Expression of the wild-type GAL4 protein is sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the co-expression of the avian c-Rel protein and its Ik B-a counterpart, p40, as GAL4 fusion proteins. Restoration of growth sensitivity towards cycloheximide requires the association of c-Rel and p40 at the GAL1 promoter and correlates with the ability of the c-Rel/p40 interaction to activate expression from the GAL1 promoter Another reverse hybrid system makes use of the most widely used counter-selectable marker in yeast genetics, URA3, which encodes orotidine-5’-phosphate decarboxylase, an enzyme required for the biosynthesis of uracil. Ye