Strong transcriptional activators isolated from viral DNA by the ‘activator trap', a novel selection system in mammalian cells

摘要

Transcription factors often contain activation domains that interact with the basic transcription machinery. We have developed a functional screening strategy in mammalian cells to selectively isolate activation domains from a library of random DNA inserts. For this, sonicated DNA fragments are cloned next to the DNA binding domain of GAL4 factor in a plasmid that also contains the SV40 origin of replication. Pools of fusion protein clones are transfected into CV-1-5GT monkey cells containing an SV40 T antigen gene under the control of a promoter with GAL4 binding sites. Plasmids that express functional transactivating fusion proteins activate the T antigen gene, thus promoting selective amplification of the plasmid in the mammalian host cell line. Using this method, we were able to select strong enhancer-type activation domains from the immediate early regions of two herpesviruses, namely pseudorabies virus and bovine herpesvirus 1. In both cases, the activation domains selected were homologues of the ICP4 regulatory protein of herpes simplex virus. The activation domain from pseudorabies virus is four times stronger than the activation domain of herpes simplex virus protein VP16 (Vmw65), making it the strongest activation domain characterized so far. This activator trap method should be useful for precisely localizing activation domain(s) in known factors, or to identify mammalian transcriptional adaptors that do not bind DNA and which may escape conventional detection methods.