Developmental Analysis of a Putative Juvenile Hormone Receptor

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Juvenile hormone (JH) is critical for insect development and reproduction. Although numerous whole-organismal studies of JH have been carried out, little is understood of the molecular action of this hormone. Using a genetic approach, this PI has been studying how JH acts at the molecular level in Drosophila melanogaster. Previous work led to the isolation of a mutant, Methoprene tolerant (Met), which has high resistance to JH or to methoprene, a JH insecticidal analog. Met flies were shown to have an intracellular JH binding protein, perhaps a JH receptor, possessing a reduced affinity for JH III. There is good evidence that this change in affinity is responsible for the resistance. Both the phenotype of Met resistance and the JH binding data suggest that the Met+ gene may encode the receptor. The DNA region containing the Met+ gene has been cloned, two transcripts have been identified, and cDNA molecules have been isolated and 80% sequenced. The work is now poised for a molecular developmental study of this gene and its product. Met+ cDNAs will be expressed to generate both fusion and non-fusion Met+ protein. Met+ fusion protein will be used to raise a polyclonal antibody specific for Met+ protein. This antibody will be used to identify Met+ protein in biochemical preparations as well as determine its intracellular localization. Particular attention will be given to possible interaction of the antibody with polytene chromosomes to determine if the Met+ protein interacts with chromosomes, presumably as a transcription factor. Authentic Met+ non-fusion protein will be generated for each of the two Met+ full-length cDNAs following expression in a baculoviral expression system. The ability of each of these proteins to bind JH III and JH bisepoxide will be evaluated. The hypothesis that Met+ encodes a JH binding protein will be directly tested in these experiments. Finally, in work designed to bridge to future studies of JH action in Drosophila, novel methoprene-resistant mutations on the second and third chromosomes will be isolated following FRT- FLP screening. The screen will be designed to identify loci whose gene product may act independently or may physically interact with the Met+ protein to confer resistance. This work will have significance in several ways: (1) the intracellular localization and JH binding of the Met+ gene product will be evaluated, leading to a better understanding of JH action; (2) novel loci involved in JH action may be identified; and (3) since Met mutants are resistant to JH insecticidal analogs, a better understanding of this resistance mechanism to these compounds will be gained. This knowledge may be applicable for resistance management of pest insects under control with JH insecticidal analogs.