生物学模式生物酵母研究入门指导.pptVIP

Getting further: two-hybrid system The yeast two hybrid system is a method to detect the interaction of two proteins in the yeast cell and it can be used to select for an interacting partner of a known protein The original version uses a transcriptional read-out to monitor interaction, nowadays there are also other methods The method is so powerful since it is not restricted to yeast proteins; the interacting partners can origin from any organism; in fact some versions do not use any yeast sequences Basis for the system is the modular nature of transcription activators that consist of exchangeable DNA binding and transcriptional activation domains lexA site reporter The gene of interest, the bait, is cloned in fusion with a DNA binding domain, such as that of the E. coli lexA protein The potential binding partner, the target or prey, which may be a library, is cloned in fusion to a transcriptional activation domain, such as that from VP16, a viral protein Only when bait and target interact, a reporter gene whose only promoter is a lexA binding site will be activated Application of the yeast two-hybrid system The possible applications to the two-hybrid system are absolutely tremendous The system can be used to detect interaction between two proteins The system can be used to characterise the domains and residues in the two proteins that mediate interaction; this can be done by mutagenesis and the use of a counterselectable reporter, such as URA3 The system can of course be used to find interaction partners The system can be used to find proteins that regulate the interaction between two proteins The system can be used to screen for drugs that inhibit the interaction between two proteins The system is actually used to construct an genome-wide map of protein interactions in yeast; using laboratory robots 6000 bait strains are crossed to 6000 prey strains to study all possible protein intercations etc Genetic analysis in action: the HOG pathway The analysis of the osmos