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Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B
S M Ruben1, P J Dillon, R Schreck
1Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110-1199.
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-kappa B 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 kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex.
Insights
Researchers identified a novel 65-kilodalton protein in human lymphocytes. This protein, related to the c-rel proto-oncogene, may be the p65 subunit of the active Nuclear Factor kappa B (NF-kappa B) transcription factor complex.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- The proto-oncogene c-rel, Drosophila dorsal, and NF-kappa B p50 subunit share conserved domains.
- NF-kappa B is a crucial transcription factor in immune responses.
Purpose of the Study:
- To identify and characterize novel rel-family proteins involved in NF-kappa B signaling.
- To investigate the role of a newly identified rel-related protein in T and B lymphocytes.
Main Methods:
- Polymerase chain reaction (PCR) and degenerate oligonucleotides were used to generate a DNA probe.
- Complementary DNA (cDNA) hybridization identified a 2.6-kilobase messenger RNA (mRNA) in lymphocytes.
- In vitro transcription and translation produced a 65-kilodalton (kD) protein for functional analysis.
Main Results:
- The 65-kD protein exhibited weak DNA binding specific for the kappa B motif.
- Binding was inhibited by I kappa B-alpha and -beta proteins, similar to the p65 NF-kappa B subunit.
- The 65-kD protein associated with the NF-kappa B p50 subunit, forming a complex with identical electrophoretic mobility to the NF-kappa B-DNA complex.
Conclusions:
- The identified 65-kD rel-related protein is likely the p65 subunit of the active NF-kappa B transcription factor.
- This finding contributes to understanding the composition and regulation of the NF-kappa B complex.