RESUMEN
The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage.
Asunto(s)
Dípteros/crecimiento & desarrollo , Dípteros/genética , Ecdisterona/metabolismo , Genes de Insecto , Proteínas de Insectos/genética , Proteínas y Péptidos Salivales/genética , Animales , Dípteros/metabolismo , Glándulas Endocrinas/metabolismo , Amplificación de Genes , Proteínas de Insectos/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas y Péptidos Salivales/metabolismoRESUMEN
Recently we have shown that BhSGAMP-1 is a developmentally regulated reiterated gene that encodes an antimicrobial peptide (AMP) and is expressed exclusively in the salivary glands, at the end of the larval stage. We show, for the first time, that a gene for an AMP is directly activated by 20-OH ecdysone. This control probably involves the participation of short-lived repressor(s). We also found that the promoter of BhSGAMP-1 is not equipped with elements that respond to infection, provoked by the injection of microorganisms, in the salivary glands or in the fat body. We produced polyclonal antibodies against the synthetic peptide and found that the BhSGAMP-1 peptide is secreted in the saliva. The BhSGAMP-1 gene was also activated during the third larval molt. These facts confirm our hypothesis that this preventive system of defense was selected to produce an environment free of harmful microorganisms in the insect's immediate vicinity, during molts.
Asunto(s)
Dípteros/genética , Ecdisterona/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas de Insectos/genética , Glándulas Salivales/efectos de los fármacos , Proteínas y Péptidos Salivales/genética , Animales , Western Blotting , Dípteros/metabolismo , Dípteros/microbiología , Ecdisterona/fisiología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/fisiología , Interacciones Huésped-Patógeno , Proteínas de Insectos/metabolismo , Larva/efectos de los fármacos , Larva/genética , Larva/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/fisiología , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Staphylococcus aureus/fisiologíaRESUMEN
Gene amplification occurs in Bradysia hygida salivary glands, at the end of the fourth larval instar. The hormone 20-hydroxyecdysone (20E) triggers this process, which results in DNA puff formation. Amplified genes are activated in two distinct groups. The activity of the first group is dependent on high levels of 20E, while the second group needs low hormone levels. Consequently, the salivary glands of B. hygida constitute an interesting biological model to study how 20E, and its receptors, affect gene amplification and activity. We produced polyclonal antibodies against B. hygida EcR (BhEcR). In western blots a polypeptide of about 66 kDa was detected in salivary gland extracts. The antibodies were also used for indirect immune-localization of BhEcR in polytene chromosomes. RNA-polymerase II was also immune-detected. We did not detect the receptor in chromosome C where the first and second groups of DNA puffs form during DNA puff anlage formation, but it was present during puff expansion. During the active phase of both groups of DNA puffs, RNA polymerase II co-localized with BhEcR. After puff regression, these antigens were not detected. Apparently, EcR plays a direct role in the transcription of amplified genes, but its role in gene amplification remains enigmatic.
