RESUMEN
The intracellular target of diphtheria toxin is a modified histidine residue, diphthamide, in the translation elongation factor, eEF2 (also known as EFT1). This enigmatic modification occurs in all eukaryotes and is produced in yeast by the action of five gene products, DPH1 to DPH5. Sequence homologues of these genes are present in all sequenced eukaryotic genomes and, in higher eukaryotes, there is functional evidence for DPH1, DPH2, DPH3 and DPH5 acting in diphthamide biosynthesis. We identified a mouse that was mutant for the remaining gene, Dph4. Cells derived from homozygous mutant embryos lacked the diphthamide modification of eEF2 and were resistant to killing by diphtheria toxin. Reporter-tagged DPH4 protein localized to the cytoskeleton, in contrast to the localization of DPH1 and consistent with evidence that DPH4 is not part of a proposed complex containing DPH1, DPH2 and DPH3. Mice that were homozygous for the mutation were retarded in growth and development, and almost always die before birth. Those that survive long enough had preaxial polydactyly, a duplication of digit 1 of the hind foot. This same defect has been seen in embryos that were homozygous for mutation of DPH1, suggesting that lack of diphthamide on eEF2 could result in translational failure of specific proteins, rather than a generalized translation downregulation.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Desarrollo Embrionario , Histidina/análogos & derivados , Factor 2 de Elongación Peptídica/metabolismo , Animales , Cromosomas de los Mamíferos/genética , Cruzamientos Genéticos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Toxina Diftérica/farmacología , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Proteínas del Ojo/genética , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Pruebas Genéticas , Genotipo , Histidina/metabolismo , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Mutantes , Mutación/genética , Células 3T3 NIH , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Empalme del ARN/efectos de los fármacos , Proteínas Represoras/genética , Dedos del Pie/anomalíasRESUMEN
Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-mediated coat color mutant white-based brown (B(w)). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis.
