RESUMEN
As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli ß-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.
Asunto(s)
Aedes/genética , Animales Modificados Genéticamente/genética , Cuerpo Adiposo/fisiología , Proteínas de Insectos/genética , Larva/genética , Región de Flanqueo 5' , Animales , Elementos de Facilitación Genéticos/genética , Escherichia coli , Femenino , Regulación de la Expresión Génica , Masculino , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , beta-Galactosidasa/genéticaRESUMEN
Chromophore substrates Dnp-Ala-Glu-Phe-Ala-Arg-NH2 and Dnp-Ala-Ala-Phe-Nle-Ala-Arg-NH2 of aspartic proteases were synthesized by a combination of chemical and enzymic methods. The kinetic parameters of their hydrolysis with pepsin, aspergyllopepsin, and chymosin were determined. The introduction of Nle in the P1' position gives stable enzyme-substrate complexes with pepsin and chymosin. A Glu residue at the P2 position contributes significantly to an increase in kcat for the chymosin hydrolysis.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Compuestos Cromogénicos/síntesis química , Compuestos Cromogénicos/metabolismo , Hidrólisis , Cinética , Especificidad por SustratoRESUMEN
Chromogenic hexapeptides Dnp-Ala-Ala/Ser-Phe-Phe-Ala-Arg-NH2 containing a Phe-Phe bond, which is sensitive to aspartic proteinases, were used as substrates for assaying the activity of pepsin, chymosin, and aspergillopepsin A. The assay was performed after the separation of hydrolyzates on SP-Sephadex by measuring at 360 nm the absorbance of the dinitrophenylpeptide lacking the cationic group, which was formed upon the cleavage of the substrate. The kinetic parameters of the hydrolysis of the substrates were evaluated. It is shown that replacing the Ala residue with Ser in the P2 position does not substantially change the kinetic parameters. The substrates were hydrolyzed by pepsin several times faster than by aspergillopepsin A or chymosin. The method is sensitive and enables the activity of aspartic proteinases to be determined easily.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Compuestos Cromogénicos/química , Oligopéptidos/química , Alanina/química , Quimosina/química , Dinitrofenoles/química , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Pepsina A/química , Serina/química , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
A general method was developed for the synthesis of new chromogenic substrates of aspartyl proteases: Dnp-Ala-Xaa-Phe-Phe-Ala-Arg-NH2, where Xaa was Ala or Ser. The synthetic scheme involved both chemical and enzymic stages, the condensation of tripeptides in an organic medium by means of pepsin immobilized on Celite being among the latters. The influence of organic solvents, reaction time, and the composition and ionic strength of the buffers used in the reaction mixture and at the pepsin immobilization step on the efficacy of the pepsin-catalyzed synthesis was studied.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Compuestos Cromogénicos/síntesis química , Alanina/química , Tampones (Química) , Catálisis , Tierra de Diatomeas/química , Enzimas Inmovilizadas , Concentración Osmolar , Pepsina A/química , Serina/química , Solventes/química , Especificidad por SustratoRESUMEN
A portion of the 5'-flanking region of the female-specific hexamerin gene, Hex-1.2, from the mosquito Ochlerotatus atropalpus was used to drive expression of the luciferase reporter gene in Drosophila melanogaster. The proximal 0.7 kb of 5'-flanking DNA were sufficient to partially repress reporter gene activity in males and to drive tissue- and stage-specific expression comparable with that of the endogenous O. atropalpus Hex-1.2 gene. The Drosophila doublesex transcription factor (DSX), expressed in Escherichia coli, bound putative DSX sites of the Hex-1.2 gene differentially in vitro. Blocking expression of the female isoform of the Doublesex transcription factor in transgenic female flies resulted in reduction of luciferase expression to levels comparable with those in males, suggesting that Doublesex could contribute to regulation of female-specific expression of the O. atropalpus Hex-1.2 gene.