REGULATION OF A DEVELOPMENTALLY CRITICAL INSECT PROTEIN

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The objective of the proposed grant is to use the differential mechanisms of control of a developmentally regulated protein as study of hormonal regulation of development. The protein, juvenile hormone esterase (JHE), is subject to neurohormonal regulation at one time during development and then later by its own substrate. Thus, the system is attractive for a comparative study on how JH acts to regulate gene expression, and how the same gene can be regulated by chemically unrelated hormones. Since a number of medically important insects and other invertebrates have been shown to possess, or respond to, juvenile hormone, the results of the proposed study may identify new approaches to the control of these organisms. The specific aims of the proposed study are: 1) provide the developmental profile of JH esterase and its electrophoretic and kinetic forms, both in their site of synthesis (fat body) and in the hemolymph, 2) determine the N-terminus sequence and other biochemical characteristics of the protein, 3) test for the biochemical level at which the hormones regulate JHE, 4) prepare cDNA clones for the protein(s) to use in measuring changes in mRNA levels of the protein during normal development and following hormonal manipulation. Both in vitro fat body and in vivo procedures will be used. Enzymatic and immunological methods will be used to measure the normal changes in the electrophoretic and kinetics forms of JHE. The electrophoretic forms will be separately purified for N- terminus sequencing and biochemical characterization. The N- terminus sequence will be used to synthesize an oligonucleotide probe, with which clones for the enzyme will be screened and identified. This probe, and the immunological and enzymatic techniques, will be used to test for the action of the neurohormonal factor, and juvenile hormone, on the following biochemical levels of regulation: changes in mRNA abundance, changes in level of translatable mRNA, change in amount of inactive and active JHE in the fat body and rate of release from the fat body, change in JH esterase turnover.