返回Google圖書搜尋

Yeast Two-hybrid Interaction Partner Screening Through in Vivo Cre-mediated Binary Interaction Tag Generation

出版	State University of New York at Buffalo, 2007
URL	http://books.google.com.hk/books?id=dEQ1tAEACAAJ&hl=&source=gbs_api

註釋Yeast two-hybrid (Y2H) is the tool of choice for the identification of novel protein-protein interactions, and it has been successfully used for genome-wide screens with several model organisms. Nonetheless, the logistics of pair-wise screening has limited its application to complex genomes. Here a modification of Y2H is developed that eliminates the requirement for pair-wise screening. This is accomplished by incorporating lox sequences into yeast two-hybrid vectors such that cDNAs encoding interacting partners become physically linked in the presence of Cre recombinase in vivo . Once linked, DNA from complex pools of clones can be processed without losing the identity of the interacting partners. Short linked sequence tags from each pair of interacting partners (B inary I nteraction Tags or BI-Tags) are then recovered using a type IIS restriction endonuclease (MmeI) and sequenced. To validate the approach, a comparison between interactions found using traditional Y2H and the BI-Tag method was performed to identify interaction partners for mouse HoxA1-DBD fusion protein from an activation-domain cDNA library made from 12.5 dpc whole mouse embryo poly A+ RNA. The BI-Tag method yielded similar results to that of traditional Y2H screening but with a significant improvement in efficiency. Several promising interaction partners for HoxA1 including Uhrf1 were identified. A second BI-Tag yeast two-hybrid screen was conducted with a library of DBD-cDNA fusion proteins for interaction partners from an AD-cDNA library, both made from 12.5 dpc whole embryo poly A+ RNA. Performing BI-tag Y2H on a library by library screen further proves the utility of the method for high-throughput Y2H. These data demonstrate that the BI-Tag technology accurately represents the complexity of the interaction partners found in the screen. The technology described here sufficiently improves the throughput of the Y2H approach to make feasible the generation of near comprehensive interaction maps for complex organisms.