The long term objective of this study is to determine the molecular mechanisms by which the juvenile hormone esterase gene from Trichoplusia ni is precisely regulated to be expresssed during the prepupal stage of metamorphosis. This competitive renewal is for the second part of an originally proposed 5 year research project. In funding the initial part, the Study Section previously determined that the specific aims of the original 5 year proposal should be separated into two sequential grants, the first grant being the presently expiring 3 year study on the original specific aims 1 and 2. Our studies on the first part have progressed nicely on track, with the unexpected discovery that the JH esterase gene is apparently organized as a more complex composite core promoter, and functions so in both lepidopteran and mosquito C7-10 cells. We describe here the studies for the remaining funding of the second part, concerning the original specific aim 3 (isolation of the proteins) and specific aim 4 (cloning of the proteins) of the original proposal, as well as the next initial characterization of these molecules that confer the to the core promoter its specific attributes as composite core promoter. Thus, the specific aims of the present proposal are:
Specific Aim 1 - Isolation of the protein(s) binding to the important, transcriptionally active promoter motifs, using protein fractionation and ligand-binding methods (specific aim 3 of original 5 year proposal) Specific Aim 2 - Cloning of these isolated transcription factors from T. ni and use of these clones to isolate the corresponding clones from mosquito cDNA libraries (specific aim 4 of original 5 year proposal) Specific Aim 3 - Functional characterization of the factors cloned from T. ni and mosquitos in their respective cell lines, and for developmental-, tissue and hormonal specificities The NIH mission in Tropical Medicine and Parasitology supports experimental projects designed toward study and control of insect disease vectors. The proposed research on much understudied composite promoters of genes whose precise control is especially crucial will identify for the first time the diagnostic component and properties of such promoters for their identification and disruption in mosquitoes and other medically important insects.
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