Molecular Analysis of Eclosion Hormone in the Tobacco Hornworm

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This project will examine the molecular structure and the regulation of synthesis of the neuropeptide, eclosion hormone (EH), in the tobacco hornworm, Manduca sexta. EH is synthesized both in the brain and in the ventral CNS and released before each molt to elicit a preprogrammed pattern of neural activity culminating in ecdysis. The 62 amino acid peptide has recently been sequenced and we intend to isolate the EH gene using synthetic oligonucleotide probes designed based on the protein sequence. The isolation of the EH gene will offer the opportunity to study the synthesis and action of a neuropeptide in the relatively simple nervous system of an insect with it's identifiable neurons. We will examine the structure of the EH gene by a combination of nucleic acid sequencing and transcript mapping studies and will identify other potential peptides derived from the prohormone. We will determine the developmental regulation of EH mRNA synthesis and will analyze the endocrine factors that regulate its expression. The cellular localization of the EH transcript will be determined by in situ hybridization both in the brain and in the cells associated with the ventral CNS, the two sources of EH. Finally, the presence of EH-like sequences in other insects will be examined by hybridization to the EH gene.