ABSTRACT
The biological activity of the transcription factor NF-kappaB is differentially controlled by three IkappaB proteins, Ikappa Balpha, Ikappa Bbeta, and Ikappa Bepsilon. We have examined the molecular basis for the differential inhibitory strengths of IkappaB proteins by constructing hybrid IkappaBs and found that the first ankyrin repeat of Ikappa Balpha is responsible for its strong inhibitory effect. Swapping a putative beta-turn within the first ankyrin repeat between the strong Ikappa Balpha and the weak IkappaBbeta inhibitors switches their in vivo inhibitory activity on NF-kappaB. The qualitatively distinct contacts made by this beta-turn in Ikappa Balpha and Ikappa Bbeta with NF-kappaB determine the efficiency by which IkappaBs sequester NF-kappaB to the cytoplasm, thus explaining their distinct effects on gene activity.
Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Proteins , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Ankyrins , DNA-Binding Proteins/genetics , Models, Molecular , Molecular Sequence Data , NF-KappaB Inhibitor alpha , Protein Binding , Protein Structure, Secondary , Proto-Oncogene Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Amino AcidABSTRACT
Using deficiencies from Drosophila melanogaster, we looked for genomic regions in the sister species D. simulans that could cause lethality when hemizygous on a hybrid genetic background. Such genotypes allow hemizygous genes from one species to interact with heterozygous genes from other species and may correspond to the kinds of genotypes causing Haldane's rule, the observation that if only one gender is sterile or inviable in species hybrids, it is nearly always the heterogametic sex. A survey of roughly 50% of the D. simulans genome (114 chromosome regions) revealed only four regions causing hybrid lethality and five causing severe reductions in hybrid viability. However, the viability of all of these genotypes was at least partially restored by rearing hybrids at lower temperature or using different genetic backgrounds from D. simulans. We therefore detected no D. simulans chromosome regions causing unconditional hybrid lethality, although several regions were shown to be deleterious under most tested temperatures and genetic backgrounds. The relative paucity of "inviability genes" supports the idea, suggested by work on other species, that hybrid inviability between closely related species might be caused by interactions among relatively few genes, while hybrid sterility may involve many more loci.
Subject(s)
Drosophila/genetics , Genes, Insect , Genome , Animals , ChimeraABSTRACT
The biological activity of the transcription factor NF-kappaB is mainly controlled by the IkappaB proteins IkappaBalpha and IkappaBbeta, which restrict NF-kappaB in the cytoplasm and enter the nucleus where they terminate NF-kappaB-dependent transcription. In this paper we describe the cloning and functional characterization of mouse IkappaBepsilon. Mouse IkappaBepsilon contains 6 ankyrin repeats required for its interaction with the Rel proteins and is expressed in different cell types where we found that it is up-regulated by NF-kappaB inducers, as is the case for IkappaBalpha and human IkappaBepsilon. IkappaBepsilon functions as a bona fide IkappaB protein by restricting Rel proteins in the cytoplasm and inhibiting their in vitro DNA binding activity. Surprisingly, IkappaBepsilon did not inhibit transcription of genes regulated by the p50/p65 heterodimer efficiently, such as the human interferon-beta gene. However, IkappaBepsilon was a strong inhibitor of interleukin-8 expression, a gene known to be regulated by p65 homodimers. In addition, IkappaBepsilon appears to function predominantly in the cytoplasm to sequester p65 homodimers, in contrast with the other two members of the family, IkappaBalpha and IkappaBbeta, which also function in the nucleus to terminate NF-kappaB-dependent transcriptional activation.