GENETICS OF A HORMONE-RESISTANT DROSOPHILA MUTANT

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Methoprene, a chemical analogue of juvenile hormone (JH), is toxic when topically applied to Drosophila melanogaster. We are examining mutants of Drosophila which are resistant to toxic dose of methoprene, and we have identified a locus which confers as much as 100-fold resistance to exogenous methoprene or JH III. This locus, Met, has been genetically and developmentally characterized. We believe that there is statistical indirect evidence that Met controls reception of JH in target tissues, possibly through a JH- receptor protein. We also believe that this locus is of sufficient importance to warrant additional biochemical and genetic analysis, which is outlined in three phases in this proposal. 1). A direct determination of Met control of a JH receptor protein will be carried out by measuring 3H-JH III binding capacity and binding affinity in cytosolic extracts from hemolymph-free larval epidermal as well as adult vitellogenic ovary tissue. We expect to see a clear difference between wild-type and mutant 3H-JH III binding characteristics if Met controls a JH receptor protein. 2). Additional alleles of Met will be recovered following EMS and gamma-ray mutagenesis, and the phenotypic characteristics of each allele will be evaluated. We will allow for the recovery of both lethal and/or female-sterile alleles in the screen as well as temperature-sensitive alleles. These alleles will be useful not only for identifying cloned Met+ sequences but also for uncovering novel roles for JH in Drosophila from an examination of the spectrum of Met phenotypes, presumably reflecting the consequences of aberrant JH binding to target tissues. 3). Finally, Met+ DNA sequences will be cloned so that we can reconstruct the MET+ gene and flanking sequences. We have selected Met alleles from a hybrid-dysgenic cross and expect these alleles to have resulted from P-element mutagenesis. After confirming this by in situ hybridization, we will use a P-element probe to identify Met sequences from a genomic library constructed from DNA from flies carrying one of the hybrid dysgenic alleles. A Met sequence restriction fragment will then be used to probe a Met+ library, and overlapping sequences isolated and aligned to reconstruct Met+. Probes from Met+ will be used to study Met+ poly(A)+ transcripts during Drosophila development.