The first developmental choice faced by germ cells in the majority of higher eukaryotes is oogenesis versus spermatogenesis, but very little is known about how this basic event is genetically encoded in any organism. We have chosen to study the developmental genetics of this germline sex determination process in the fruit fly, Drosophila melanogaster. Our long-term goals are to: 1) identify the major germline sex determination regulatory genes, 2) determine how they are related in terms of a developmental hierarchy and 3) understand in detail how these regulatory gene products function. We are question oriented and take advantage of genetic, molecular, genomic, and biochemical techniques. Current molecular studies are focused on OVO, a highly conserved zinc-finger domain protein functioning during germline development in insects and mammals. A standard model for regulated transcription holds that regulatory proteins bind enhancer and silencer sequences and influence the expression from a basal core promoter bound by ubiquitous transcriptional machinery. Interestingly, OVO protein binds to an enhancer and to a core promoter element of the downstream ovarian tumor locus. We have now definitively shown that both the upstream enhancer and the core promoter sites are functional by using reporter genes. We suggest that OVO acts as a tissue-specific transcription factor at both the expected enhancer location and at the core promoter. Only core promoters with OVO binding sites will respond to OVO enhancers. We are looking for other components that act in conjunction with OVO at the transcription initiation site. OVO can act as both a transcriptional activator and as a transcriptional repressor at ovarian tumor. We have shown that two families of OVO proteins, OVO-A and OVO-B (positive) and OVO-A (negative) have opposing activities in flies, both in terms of transcription and in terms of phenotypes. Interestingly, OVO-A and OVO-B have thematically similar opposing activities in yeast one-hybrid experiments, suggesting that OVO proteins interact with transcriptional components conserved in yeast. Future work on OVO will be focused on how OVO-B versus OVO-A isoforms are regulated and on the downstream targets of OVO function. We are developing a gonadogenesis DNA microarray to aid our studies on OVO function. The first step, development of testis EST and a testis subarray have been completed. We are now adding ovarian ESTs to the array and have begun experiments to look for genes differentially regulated by OVO-A and OVO-B