Identification and characterization of steroid regulated genes in Manduca sexta CNS.
Committee ChairMorton, David B.
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PublisherThe University of Arizona.
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AbstractThe major developmental cue and the coordinating factor of metamorphic changes in insects is a group of steroid hormones, the ecdysteroids. Using subtractive hybridization I have isolated five cDNA clones that represent genes whose transcripts are up-regulated prior to pupal ecdysis in the nervous system of the tobacco hornworm, Manduca sexta, after a major ecdysteroid peak. Three of the genes, Mng10, Mng14 and tps9 are expressed primarily in the nervous system, the two other genes, esr16 and esr20 are specific to tracheal epithelial cells. In situ hybridizations showed that the transcripts of the two nervous system specific genes were localized to uniquely identifiable neurosecretory cells in the brain and ventral nervous system. The total number of cells expressing these genes in the CNS is 16-20. The third nervous system specific transcript is localized to small patches along the boundary between the central neuropil and neuronal cell bodies, in abdominal ganglia. The patches are smaller than nuclei, possibly mitochondria, and seem to be localized to large tracheal branches. Sequence analysis of the cDNA clones revealed that esr20, esr16 and Mng10 encoded a protein, with slight similarity to already identified sequences. These similarities, however, were insufficient to propose a function for the Manduca genes. Two of the sequences do not seem to encode a protein and are postulated to function as regulatory or structural RNAs. Statistical analysis of the coding and non-coding sequences showed a clear distinction between their behavior using the Zipf'test. Four of the five genes are regulated by ecdysteroids as predicted by the initial screen, transcript levels are reduced at high 20-HE concentrations. More detailed studies of the regulation of a tracheal-specific gene (esr20) showed that the accumulation of the mRNA requires first the presence then the subsequent absence of the ecdysteroids. The role of the ecdysteroids appears to be permissive and an additional factor is required to initiate the accumulation of the mRNA. The transcript of esr20 appears 16 hours before ecdysis and is removed at ecdysis by a mechanism that requires protein synthesis and possibly influences the stability of the transcript.