9316896 Henrich Steroidogenesis is a developmentally regulated, neurohormonally stimulated, second messenger mediated process whose molecular regulation remains largely undefined in a variety of experimental systems. Among insects, neural factors periodically stimulate the synthesis of the insect steroid hormone, ecdysone, in the larval prothoracic gland. This steroid is then converted into its active form, 20- hydroxyecdysone, and interacts with nuclear receptors in target tissues to trigger the developmental changes associated with larval molts and metamorphosis. This project will identify and characterize several genes at the molecular level suspected to be involved in the regulation of ecdysteroid biosynthesis in a model organism, the fruit fly, Drosophila melanogaster. Specifically, the project will pursue the following objectives; 1) clone a gene by chromosomal walking that is defined by a conditional mutation (ecdysoneless; ecd-1) which in turn, causes an ecdysteroid deficiency at the mutation's restrictive temperature; 2) deduce the structure of the ecd gene product from cDNA clones, delineate its developmental expression, assess its functional role in steroidogenesis, and construct a physical map of the gene; 3) identify and characterize members of the steroid hormone receptor super family active in Drosophila steroidogenic tissues, including those whose mammalian homologues have already been implicated in steroidogenic regulation and those implicated in ecdysteroid regulation. As part of achieving the first objective, a cDNA library will be prepared by reverse transcriptase-polymerase chain reaction methods with RNA extracted from the Drosophila ring gland, which contains the prothoracic gland. These methods will also be employed to quantify receptor transcripts through the developmental period during which the capacity of the Drosophila larval prothoracic gland to produce ecdysteroids and respond to proteinaceous neural factors increases dra matically. By achieving the aims of this project, a foundation will be laid for future genetic investigations in a powerful model system concerning the individual roles and functional coordination of molecular activities that underlie steroidogenesis, as well as the relationship between steroidogenic activities and steroid dependent activities in target tissues. ***
