Molecular Basis of Retrotransposon Mobilization

描述（由申请人提供）：本申请的目标是 确定逆转录病毒插入特异性的分子基础。吉普赛 果蝇的逆转录病毒优先插入位于5'端的区域， 卵子基因的终结该区域包含Ovo蛋白的结合位点， 这表明卵基因的产物，可能是OvoA，可能在 过程为了验证这一假设，我们将突变这些卵结合位点， 并且将检查对吉普赛插入的影响。一个碎片， 将分析串联重复的合成Ovo结合位点以确定 是否会导致吉普赛人频繁插入如果OvoA蛋白 负责插入特异性，它可能通过与 吉普赛人编码的蛋白质我们将使用生物化学方法来确定 吉普赛蛋白与OvoA相互作用。一旦这种蛋白质被鉴定出来，我们将 分析参与这种相互作用的OvoA结构域。然后我们将融合 将该OvoA结构域与GAL 4的DNA结合结构域结合，并确定DNA 含有GAL 4结合位点的片段可以诱导高频率的吉普赛人 动员。如果OvoA-GAL 4融合蛋白能够引起高频率的 吉普赛人的融合，我们将试图利用这一观察， 识别被特定DNA结合蛋白激活或抑制的基因。我们将 构建携带编码Hunchback-OvoA融合基因的转基因果蝇 蛋白吉普赛人的动员应该引起新的插入， Hb-OvoA蛋白靶向基因调控区的元件 其表达受Hb如Kruppel和其它gap基因调控。 这些实验的结果将使我们能够了解 逆转录病毒插入特异性在足够的分子细节， 能够在受控条件下操纵吉普赛人的动员， 直接将吉普赛人的插入定位到特定的位点。这些结果也将有助于 了解果蝇维持反转录转座子的机制 动员，同时确保最小的有害影响。

DESCRIPTION (provided by applicant): 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.