The goal of this application is to determine the molecular basis for retroviral insertional specificity. The gypsy retrovirus of Drosophila inserts preferentially into a region located in the 5' end of the ovo gene. This region contains binding sites for the Ovo proteins, suggesting that products of the ovo gene, perhaps OvoA, might play a role in the process. To test this hypothesis, we will mutate these Ovo binding sites and the effect on gypsy insertion will be examined. A fragment containing tandemly repeated synthetic Ovo binding sites would be analyzed to determine whether it could cause high frequency of gypsy insertions. If the OvoA protein is responsible for insertional specificity, it might do so by interacting with gypsy-encoded proteins. We will use biochemical approaches to determine which gypsy proteins interact with OvoA. Once this protein(s) is identified, we will analyze the domains of OvoA involved in this interaction. We will then fuse this OvoA domain to the DNA binding domain of GAL4 and determine whether a DNA fragment containing GAL4 binding sites can induce high frequency of gypsy mobilization. If the OvoA-GAL4 fusion protein is able to elicit high frequency of gypsy integration, we will attempt to use this observation to try to identify genes activated or repressed by specific DNA binding proteins. We will construct transgenic flies carrying a gene encoding a Hunchback-OvoA fusion protein. Mobilization of gypsy should give rise to new insertions of this element targeted by the Hb-OvoA protein into the regulatory regions of genes whose expression is regulated by Hb such as Kruppel and other gap genes. Results from these experiments will allow us to understand the mechanisms of retroviral insertional specificity in sufficient molecular detail to eventually be able to manipulate gypsy mobilization under controlled conditions and directly target gypsy insertion to specific loci. These results will also help understand mechanisms developed by Drosophila to maintain retrotransposon mobilization while at the same time ensuring minimal deleterious effects.
