Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system.
A 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 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 activation 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 metallothionein (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.
Inouye
C
C
Department of Pharmacology, UCSD School of Medicine, La Jolla 92093-0636, USA.
Remondelli
P
P
Karin
M
M
Elledge
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015723:Gene Library; D008322:Mammals; D008668:Metallothionein / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D008668:Metallothionein