Expression Coupled Gene Activation

Another means to detect recombinant gene expression, which has recently gained substantial popularity, is the coupling of recombinant gene expression to the transactivation of another unique gene This approach, an example of which is the yeast two-hybrid system, is particularly useful for the detection of interacting proteins, and the assay is performed in vivo rather than in vitro, thus permitting the detection of such proteins in their native, biologically active state. The yeast two-hybrid system takes advantage of the fact that many eukaryotic transcription activators are made up of structurally separable and functionally independent domains. For example, the yeast transcriptional activator protein GAL4 contains a DNA-binding domain (DNA-BD), which recognizes a 17 base-pair DNA sequence, and an activation domain (AD). Upon DNA-BD binding to the specific upstream region of GAL4-responsive genes, the AD interacts with other components of the machinery to initiate transcription. Thus, both domains are needed in an interactive manner for specific gene activation to take place. In the popular yeast two-hybrid system, the two GAL4 domains are separately fused to genes encoding proteins that interact with each other, and these recombinant hybrid proteins are expressed in yeast. Interaction of the two-hybrid proteins brings the two GAL4 domains in close enough proximity to form a functional gene activator, resulting in the expression of specific reporter gene(s), thereby rendering the protein interaction, i.e , expression of the desired recombinant protein, phenotypically identifiable. In practice, the target protein gene is ligated to the DNA-BD in the form of an expression vector. The gene of interest, whose activity includes interaction with the target protein, is ligated into an AD vector. The two hybrid plasmids are then cotransformed into specialized yeast reporter strain. Expression of the desired gene thus activates a known GAL4 responsive gene(s) and confers specific phenotype to the host cell, which can be selectively identified Protocols utilizing the two-hybrid system and its variants are described in several chapters in this book (Chapter 12, 15, and 16).

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