Isolation of a rel -related Human cDNAThat Potentially Encodes the 65-kD Subunit of NF-κB

Abstract

A DNA probe that spanned a domain conserved among the proto-oncogene c- rel , the Drosophila morphogen dorsal , and the p50 DNA binding subunit of NF-κB was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel -related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the κB binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-κB subunit and binding was inhibited by IκB-α and -β proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-κB and the κB probe to form a complex with the same electrophoretic mobility as the NF-κB-DNA complex. Therefore the rel -related 65-kD protein may represent the p65 subunit of the active NF-κB transcription factor complex.