Our long term goals are directed towards understanding regulation of gene expression in the mosquito, Aedes aegypti, and developing genetic transformation of this insect. During the previous grant period we initiated an investigation of the molecular structure of two 20- hydroxyecdysone-inducible genes: the vitellogenin A1 gene, which is expressed in the fat body, and a putative vitelline membrane protein gene which is expressed in the follicle cells of the ovary. Our attention has focused on identifying regulatory sequences of these genes and developing from these sequences, chimeric constructs that can be expressed in transfected Aedes cells in culture. We have characterized the 20-hydroxyecdysone response in cultured cells from Aedes albopictus and Aedes aegypti. The identification of 20- hydroxyecdysone-inducible proteins expressed by cultured mosquito cells supports continued development of this system. We have also developed technology for expressing genes in transfected cells in response to ecdysone. Finally, we have identified in cultured cells a 25 kDa protein whose synthesis is induced by the juvenile hormone analog methoprene, and enhanced by treatment with 20-hydroxyecdysone following methoprene pre- treatment. We propose to extend these studies by: 1) continuing the primary sequence analysis of the 20-hydroxyecdysone-inducible vitellogenin Al and vitelline membrane protein genes; 2) testing whether changes in chromatin structure occur in these genes during the development of competence, and in response to hormone stimulation; 3) developing an in vitro transcription assay for analysis of the mosquito genes; 4) continuing to develop homologous transformation systems for expression of these genes in transfected cultured cells. Genetic transformation of mosquitoes is a reasonable goal given the powerful tools made available by recombinant DNA technology, and the rapid advances being made in the transformation of other organisms. Given the obvious medical importance of the mosquito, the choice of this insect as a particular target for genetic transformation is apparent.
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