Isolation of a cDNA Encoding a Metal Response Element Binding Protein Using a Novel Expression Cloning Procedure: The One Hybrid System

摘要

ABSTRACTA new method for isolation of cDNA clones encoding sequence-specific DNA-binding proteins is described. This method, the one-hybrid system, is based on the use of reporter genes whose transcription can be activated through syntheticciselements recognized by the sought-after DNA-binding protein. These reporter genes are used forin vivoscreening of a library of cDNAs fused to a DNA fragment encoding theGAL4activation domain. cDNA clones expressing the appropriate fusion proteins lead to activation of these reporter genes in transformed yeast cells. We have used this approach to isolate a mammalian cDNA clone encoding a sequence-specific DNA-binding protein that recognizes the metal response elements (MREs) of the metal-lothionein (MT) genes. The protein encoded by this cDNA, M96, shows similarity to the trithorax proteins. Expression of a functional DNA-binding form of M96 requires Zn2+ions. The recombinant protein binds to several different MREs but fails to recognize nonfunctional mutant MREs. M96 may be involved in the activation of MT genes in response to heavy-metal ions.