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 screening and isolation of cDNAs by protein-protein or protein-DNA interactions 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 illustrating the sensitivity and selectivity of the method. Using the small subunit of TFIIF (Rap30) as a target, we demonstrate the enrichment of a clone encoding the large subunit, Rap74, from a cDNA library. Additional cDNA clones including human Rap30 and an anonymous cDNA clone 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 interact not only directly with the target protein but also through bridging by endogenous proteins.
Nallur
G N
GN
Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06356.
Vasavada
H A
HA
Sankhavaram
P R
PR
Xu
W J
WJ
Weissman
S M
SM
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D005069:Evaluation Studies as Topic; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D019473:Transcription Factors, TFII; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C045264:replication initiator protein; D004247:DNA; D004265:DNA Helicases; C056604:transcription factor TFIIF