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      3. 酵母雙雜交實驗


        The establishment of yeast two-hybrid system is due to the understanding of eukaryotic cell regulation of transcription initiation.??It has been found that many eukaryotic transcriptional activators are composed of two separate and functionally independent domains.??For example, yeast's transcriptional activator GAL4 has a DNA binding domain (BD) of 147 amino acids at the N-terminal,??Terminal C has a transcription activation domain (AD) composed of 113 amino acids.??The DNA binding domain of GAL4 can bind to upstream activating sequence (UAS), while the transcriptional activation domain can activate genes downstream of UAS for transcription.??However, a single DNA binding domain cannot activate gene transcription, nor can a single transcriptional activation domain activate the downstream genes of UAS. Only when they are bound together in a certain way can they have the function of a complete transcriptional activator.??

        1 Yeast two hybrid assay process ?

        The yeast two-hybrid system takes advantage of the functional characteristics of GAL4 to capture new proteins through mutual combination of two hybrid proteins in yeast cells and transcriptional activation of reporter genes. The general steps are as follows: ?

        Treat the cDNA sequence of known protein as bait and fuse it with DNA binding domain to construct bait plasmid. ?

        The cDNA sequence of the protein to be screened was fused with the transcriptional activation domain to construct a library plasmid. ?

        The two plasmids were co-transformed into yeast cells. ?

        In yeast cells, the isolated DNA binding domain and transcriptional activation domain do not interact with each other, but the decoy protein can specifically interact with the unknown protein to activate the reporter gene transcription.??Otherwise, it cannot.??New proteins can be captured using the expression of four reporter genes.??

        2 Application

        Study the interaction between known proteins, search for domains that play key roles in protein-protein interaction, and search for new proteins that interact with target proteins.??It is believed that with the development and popularization of molecular biology technology, yeast two-hybrid technology will play a greater role in proteomics research in the future.??

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