Many cellular functions are regulated by change in genome expression. One major class of compounds which mediate these changes are hormones acting via members of the steroid/thyroid receptor superfamily. These hormone receptors alter gene expression by binding to specific regions of DNA and regulating transcription of adjacent genes. The nature of these regions of DNA and the functions of the genes regulated are of central importance to the understanding of normal physiological function, development and disease. Non-mammalian model systems have been invaluable in understanding the basic mechanisms of gene regulation by these hormones in all organisms. Studies by a number of investigators have shown the importance of using insects such as Drosophila melanogaster and Manduca sexta for such model systems. Both a rise in steroid levels and a decline can act as the signal which alters cellular function by an increase in the transcription of specific genes. The differences between the two mechanisms involved are, as yet, unclear. This project will use the insect, Manduca sexta, as a model system to characterize genes in the nervous system, whose transcription is increased by the declining levels of the steroid hormones, the ecdysteroids. Insect ecdysteroid receptors and their specific DNA binding sites have a high degree of homology with vertebrate steroid receptors, thus the mechanisms of regulation are likely to be conserved. We have isolated several genes from the nervous system of Manduca sexta whose transcripts increase in abundance as steroid levels decline. The specific steroid requirements for expression of one of these genes will be investigated with a combination of in vivo and in vitro manipulations of ecdysteroid levels. The transcription rate of this gene will be determined during development to test whether the regulation is at the transcriptional level. The effect of protein synthesis inhibitors on the levels of transcript will be determined and factors involved in the decline in transcript levels will be investigated. Studies on the regulatory regions of the gene and presence of potential regulatory proteins will be initiated. One long-term of this project is to determine the function of these genes in the development of the nervous system. An analysis of the developmental and spatial expression patterns of additional genes of this type will be completed to correlate the known physiological changes taking place in the nervous system with the presence of a particular transcript.
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