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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD024869-05
Application #
2199324
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1989-06-01
Project End
1998-03-30
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Arizona
Department
Zoology
Type
Schools of Earth Sciences/Natur
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Isoe, Jun; Hagedorn, Henry H (2007) Mosquito vitellogenin genes: Comparative sequence analysis, gene duplication, and the role of rare synonymous codon usage in regulating expression. J Insect Sci 7:1-49
Noriega, Rafael; Ramberg, Frank B; Hagedorn, Henry H (2002) Ecdysteroids and oocyte development in the black fly Simulium vittatum. BMC Dev Biol 2:6
Gerenday, A; Shih, K M; Herman, C C et al. (2001) Increased ribonucleotide reductase activity in hydroxyurea-resistant mosquito cells. Arch Insect Biochem Physiol 46:19-25
Tu, Z (1999) Genomic and evolutionary analysis of Feilai, a diverse family of highly reiterated SINEs in the yellow fever mosquito, Aedes aegypti. Mol Biol Evol 16:760-72
Shotkoski, F A; Jayachandran, G; Stamatoyannopoulos, G et al. (1999) Sequence of a mosquito ribonucleotide reductase cDNA and evidence for gene amplification in hydroxyurea-resistant cells. Insect Mol Biol 8:565-70
Shih, K M; Gerenday, A; Fallon, A M (1998) Culture of mosquito cells in Eagle's medium. In Vitro Cell Dev Biol Anim 34:629-30
Wang, Z H; Fallon, A M (1998) Similarities to a LINE element shared by Anopheline and Culicine mosquitos map to the distal end of dihydrofolate reductase amplicons in Aedes albopictus mosquito cells. Insect Biochem Mol Biol 28:613-23
Edwards, M J; Severson, D W; Hagedorn, H H (1998) Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti. Insect Biochem Mol Biol 28:915-25
Tu, Z; Isoe, J; Guzova, J A (1998) Structural, genomic, and phylogenetic analysis of Lian, a novel family of non-LTR retrotransposons in the yellow fever mosquito, Aedes aegypti. Mol Biol Evol 15:837-53
Gerenday, A; Blauwkamp, T S; Fallon, A M (1997) Synchronization of Aedes albopictus mosquito cells using hydroxyurea. Insect Mol Biol 6:191-6

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