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。吉普赛人 果蝇逆转录病毒优先插入位于5‘端的区域 卵子基因的末端。这个区域包含卵子蛋白的结合位点， 这表明卵子基因的产物，也许是卵子A，可能在 这一过程。为了验证这一假设，我们将突变这些卵子结合位点 并将考察其对吉普赛人插入的影响。包含以下内容的片段 简单重复的合成OVO结合位点将被分析以确定 是否会导致高频率的吉普赛插入术。如果卵子蛋白 负责插入的专一性，它可能通过与 吉普赛人编码的蛋白质。我们将使用生化方法来确定 吉普赛蛋白与OvoA相互作用。一旦鉴定出这种蛋白质(S)，我们将 分析参与此交互的OvoA域。然后我们将聚变 将此OvoA结构域与GAL4的DNA结合域结合，并确定是否存在DNA 含有GAL4结合位点的片段可诱发高频率的吉普赛人 动员。如果OvoA-GAL4融合蛋白能够诱导高频 吉普赛人的融合，我们将尝试利用这一观察结果来尝试 识别由特定DNA结合蛋白激活或抑制的基因。我们会 构建携带编码驼背-卵子融合基因的转基因果蝇 蛋白。吉普赛人的动员应该会带来新的插入 HB-OvoA蛋白靶向基因调控区的元件 其表达受Hb等Kruppel等GAP基因的调控。 这些实验的结果将使我们能够理解 逆转录病毒插入的特异性在足够的分子细节上最终 能够在可控的条件下操纵吉普赛人的动员和 直接将吉普赛人的插入定位到特定的基因座。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，同时确保将有害影响降至最低。

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.