C. Inouye et al., ISOLATION OF A CDNA-ENCODING A METAL RESPONSE ELEMENT-BINDING PROTEINUSING A NOVEL EXPRESSION CLONING PROCEDURE - THE ONE HYBRID SYSTEM, DNA and cell biology, 13(7), 1994, pp. 731-742
A new method for isolation of cDNA clones encoding sequence-specific D
NA-binding proteins is described. This method, the one-hybrid system,
is based on the use of reporter genes whose transcription can be activ
ated through synthetic cis elements recognized by the sought-after DNA
-binding protein. These reporter genes are used for in vivo screening
of a library of cDNAs fused to a DNA fragment encoding the GAL4 activa
tion domain. cDNA clones expressing the appropriate fusion proteins le
ad to activation of these reporter genes in transformed yeast cells. W
e have used this approach to isolate a mammalian cDNA clone encoding a
sequence-specific DNA-binding protein that recognizes the metal respo
nse elements (MREs) of the metallothionein (MT) genes. The protein enc
oded by this cDNA, M96, shows similarity to the trithorax proteins. Ex
pression of a functional DNA-binding form of M96 requires Zn2+ ions. T
he recombinant protein binds to several different MREs but fails to re
cognize nonfunctional mutant MREs. M96 may be involved in the activati
on of MT genes in response to heavy-metal ions.