RESUMEN
PURPOSE: To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS: Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS: The new mutations--ADD4802, Aey11, and Aey18--were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C-->T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G-->A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G-->A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters. CONCLUSIONS: Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.
Asunto(s)
Alelos , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Microftalmía/genética , Mutación , Factores de Transcripción Paired Box/genética , Regiones Promotoras Genéticas/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Etilnitrosourea/toxicidad , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Fenotipo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Homeobox SIX3RESUMEN
Sox2 transcription factor is expressed in neural tissues and sensory epithelia from the early stages of development. Particularly, it is known to activate crystallin gene expression and to be involved in differentiation of lens and neural tissues. However, its place in the signaling cascade is not well understood. Here, we report about the response of its promoter to the presence of other transcription factors, AP2alpha, Msx2, Pax6, Prox1 and Six3, in a transient reporter gene assay using HEK293 cells as recipient cells. Taking our data together, AP2, Pax6 and PROX1 can activate the Sox2 promoter. Msx2 has an inhibitory effect, whereas Six3 does not affect the Sox2 promoter. These data indicate a common activating cascade at least for AP2, Pax6, Prox1 and Sox2.