RESUMO
RNA helicase A (RHA) is the human homologue of the Drosophila maleless protein, an essential factor for the development of male flies. Recently, it was shown that RHA cooperates with the cAMP-responsive element in mediating the cAMP-dependent transcriptional activation of a number of genes. Due to the participation of cAMP as a second messenger in a number of signaling pathways, we examined the function of RHA during mammalian embryogenesis. To examine the role(s) of RHA in mammalian development, RHA knockout mice were generated by homologous recombination. Homozygosity for the mutant RHA allele led to early embryonic lethality. Histological analysis, combined with terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) reactions of RHA-null embryos, revealed marked apoptotic cell death specifically in embryonic ectodermal cells during gastrulation. RNA in situ analyses of the expression of HNF-3beta and Brachyury, two molecular markers for gastrulation, showed that RHA-null embryos at days 7.5 and 8.5 expressed both HNF-3beta and Brachyury in a pattern similar to those of pre- and early streak stages of embryos, respectively. These observations indicate that RHA is necessary for early embryonic development and suggest the requirement of RHA for the survival and differentiation of embryonic ectoderm.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Gástrula , RNA Helicases/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Recombinação GenéticaRESUMO
RNA helicase A is an enzyme that possesses both RNA and DNA helicase activities. In this report, we describe the isolation of a mouse cDNA encoding RNA helicase A. The deduced amino acid sequence derived from mouse RNA helicase A cDNA exhibits 87 and 47% identity to its human and Drosophila homologs, respectively. Using Southern blot analysis employing a mouse backcross panel, we have assigned the mouse RNA helicase A gene to chromosome 1, mapping near the D1Bir20 locus at MGD position 67. Northern blot and primer extension analyses indicate that, although its level is variable, RNA helicase A appears to be expressed from a single transcription start site in all tissues tested. Sequence analysis of the upstream genomic DNA revealed that the promoter region lacks a TATA box and contains two high-affinity sites for Sp1, one ISRE, a binding site for interferon regulatory factor, and three AP2-binding sites. These findings suggest that the transcriptional regulation of the RNA helicase A gene is complex.
Assuntos
DNA Complementar/isolamento & purificação , DNA/isolamento & purificação , RNA Nucleotidiltransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Éxons , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Especificidade de Órgãos/genética , RNA Helicases , RNA Nucleotidiltransferases/isolamento & purificação , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNARESUMO
The DNA-dependent protein kinase (DNA-PK) is a mammalian serine/threonine kinase that is implicated in the repair of DNA double-strand breaks, DNA replication, transcription, and V(D)J recombination. To determine the role of the DNA-binding subunit of DNA-PK in vivo, we targeted Ku80 in mice. In mutant mice, T and B lymphocyte development is arrested at early progenitor stages and there is a profound deficiency in V(D)J rearrangement. Although Ku80-/- mice are viable and reproduce, they are 40-60% of the size of littermate controls. Consistent with this growth defect, fibroblasts derived from Ku80-/- embryos showed an early loss of proliferating cells, a prolonged doubling time, and intact cell-cycle checkpoints that prevented cells with damaged DNA from entering the cell-cycle. The unexpected growth phenotype suggests a new and important link between Ku80 and growth control.
