基因研对究方法.pptVIP

* Outline of an experiment that reveals the presence of independent DNA-binding and transcription-activating domains in the yeast gene activator protein Gal4. A functional activator can be reconstituted from the C-terminal portion of the yeast Gal4 protein if it is attached to the DNA-binding domain of a bacterial gene regulatory protein (the LexA protein) by gene fusion techniques. When the resulting bacterial-yeast hybrid protein is produced in yeast cells, it will activate transcription from yeast genes provided that the specific DNA-binding site for the bacterial protein has been inserted next to them. (A) The normal activation of gene transcription produced by the Gal4 protein. (B) The chimeric gene regulatory protein requires the LexA protein DNA-binding site for its activity. Gal4 is normally responsible for activating the transcription of yeast genes that code for the enzymes that convert galactose to glucose. In the experiments shown here, the control region for one of these genes was fused to the E. coli lacZ gene, which codes for the enzyme b-galactosidase (see Figure 7-38). b-galactosidase is very simple to detect biochemically and thus provides a convenient way to monitor the expression level specified by a gene control region; lacZ thus serves as a reporter gene since it reports the activity of a gene control region. * The target protein is fused to a DNA-binding domain that localizes it to the regulatory region of a reporter gene as bait. When this target protein binds to another specially designed protein in the cell nucleus (prey), their interaction brings together two halves of a transcriptional activator, which then switches on the expression of the reporter gene. The reporter gene is often one that will permit growth on a selective medium. Bait and prey fusion proteins are generated by standard recombinant DNA techniques. In most cases, a single bait protein is used to fish for interacting partners among a large collection of prey proteins produ