EVALUATION OF THE CONTINGENT REPLICATION ASSAY (CRA) AND ITS APPLICATION TO THE STUDY OF THE GENERAL TRANSCRIPTION INITIATION-FACTOR, TFIIF

The Contingent replication assay (CRA) is a rapid assay for the screen ing and isolation of cDNAs by protein-protein or protein-DNA interacti ons in mammalian cells. The method has been shown to enrich a plasmid containing a cDNA encoding the bacterial replication-related protein, R6K, from a mixture of two plasmids. In this report we present data il lustrating the sensitivity and selectivity of the method. Using the sm all subunit of TFIIF (Rap30) as a target, we demonstrate the enrichmen t of a clone encoding the large subunit, Rap74, from a cDNA library. A dditional cDNA clones including human Rap30 and an anonymous cDNA clon e homologous to members of the human cdc2 kinase family were enriched and isolated by a modified screening approach. The structure of these additional clones suggest that the CRA enriches for products that inte ract not only directly with the target protein but also through bridgi ng by endogenous proteins.