XrelA, a Xenopus maternal and zygotic homologue of the p65 subunit of NF-kappa B. Characterisation of transcriptional properties in the developing embryo and identification of a negative interference mutant.
We have isolated two clones (XrelA.1 and XrelA.2) from Xenopus ovary representing differentially processed mRNAs homologous throughout their translated regions to the mammalian p65 subunit of NF-kappa B. The transcripts are ubiquitously present throughout development, but are most abundant in late blastulae and gastrulae. Overproduced protein shows nuclear localisation in both oocytes and early embryos. The XrelA.2 product bound to DNA as an oligomer which was not detected in the normal embryo. Two endogenous kappa B-binding complexes were present, showing no stage-specific variation, although one was relatively deficient in posterior regions of the early neurula. They were not disrupted by dimerization with over-expressed XrelA, suggesting that they were not produced by NF-kappa B/Rel/dorsal family members. The transcriptional properties of the cloned XrelA were assayed in intact embryos by co-injecting XrelA mRNA and a linear HIV LTR-driven CAT reporter gene. CAT levels were stimulated 20-30-fold by XrelA mRNA levels in the 100 pg range, and this was wholly dependent on NF-kappa B binding sites, and largely dependent on those for SP-1. These results were remarkably reproducible and show that quantitative analysis of transcription factor function is possible in intact developing Xenopus embryos A mutant lacking the transcriptional activation domain antagonised co-injected wild-type XrelA, providing a potential dominant negative p65 mutant for interfering with NF-kappa B function in analysing NF-kappa B function in normal development.
Richardson
J C
JC
Department of Biological Sciences, University of Warwick, Coventry, UK.
Garcia Estrabot
A M
AM
Woodland
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D005260:Female; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry*; D009928:Organ Specificity; D010053:Ovary / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000502:physiology; D020569:Proto-Oncogene Proteins c-rel; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D015053:Zygote / Q000737:chemistry*
D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D014157:Transcription Factors