Molecular Basis of Retrotransposon Mobilization

逆转录转座子动员的分子基础

• 批准号: 6383013
• 负责人: Victor G. Corces
• 金额: $ 26.67万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6383013
• 关键词: DNA binding protein; Drosophilidae; Retroviridae; binding sites; electron microscopy; gene expression; gene induction /repression; gene mutation; genetic transcription; host organism interaction; molecular genetics; polymerase chain reaction; protein protein interaction; transcription factor; transposon /insertion element; virus envelope; virus protein; viruslike particle

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.

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' 的区域 卵基因的末端。该区域包含 Ovo 蛋白的结合位点， 表明 ovo 基因的产物，也许是 OvoA，可能在 的过程。为了检验这个假设，我们将突变这些 Ovo 结合位点 并将检查对吉普赛人插入的影响。一个片段包含 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。我们将使用生化方法来确定哪些 吉普赛蛋白与 OvoA 相互作用。一旦识别出该蛋白质，我们将 分析参与这种相互作用的 OvoA 域。然后我们将融合 将此 OvoA 结构域与 GAL4 的 DNA 结合结构域结合，并确定是否为 DNA 含有GAL4结合位点的片段可诱发吉普赛高频 动员。如果 OvoA-GAL4 融合蛋白能够引发高频 关于吉普赛人的融合，我们将尝试利用这一观察来尝试 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 蛋白质。吉普赛人的动员应该会产生新的插入 Hb-OvoA 蛋白靶向的元件进入基因调控区域 其表达受Hb调控，如Kruppel等gap基因。 这些实验的结果将使我们能够了解其机制 逆转录病毒插入特异性具有足够的分子细节，最终 能够在受控条件下操纵吉普赛人的动员，并且 直接将吉普赛插入特定位点。这些结果也将有助于 了解果蝇开发的维持逆转录转座子的机制 动员起来，同时确保将有害影响降到最低。