Genome-wide Identification of Immediate Early Genes in Insects

描述(申请人提供)：响应神经元活动而转录的基因，称为即刻早期基因(IEGs)，包括参与重要生物学过程的成员，如突触可塑性、学习和记忆以及肿瘤发生。一些基因已经在哺乳动物中被鉴定为IEGs，但对于昆虫中的IEGs却知之甚少，例如被用作研究这些生物过程的模型的黑腹果蝇。此外，人们对神经元活动下调的基因知之甚少。我们建议进行全基因组转录组分析，以确定在三种昆虫中，包括黑腹果蝇、拟黑腹果蝇和疟疾媒介冈比亚按蚊的感觉神经元活性上调和下调的基因。通过对三个不同物种进行这一分析，我们将能够确定每个物种的候选IEG，以及识别在进化上保守的IEG，这些IEG在三个物种中受到类似的调节。我们建议使用一种成熟的定量RT-PCR方法来验证识别的候选IEG。最后，我们建议使用两种替代策略，RNA原位分析和转基因IEG启动子分析，将IEG活性诱导转录的特异性映射到适当的神经元。对于转基因策略，我们建议识别IEGs的候选调节区，并在转基因果蝇中测试这些启动子序列，以确定它们是否能够驱动报告基因的表达，以响应神经元的活动。拟议研究的成功完成将提供一个全基因组的基因目录，这些基因在果蝇和按蚊中以依赖活动的方式进行调控。一种成功的转录报告昆虫体内神经元活动的方法将是一个巨大的进步，并可能适用于复杂的神经生物学问题的研究，包括化学感觉信息的高级加工、化学感觉编码、感觉整合和记忆形成。 与公共健康相关：我们建议使用全基因组的方法来确定进化上保守的基因，这些基因受果蝇和按蚊神经元激活的调控。在哺乳动物中，活动调节基因与神经系统功能和癌症生物学等许多重要的生理过程有关，在模式昆虫黑腹果蝇中研究它们的能力将有助于揭示它们调节和功能的基本机制。这些基因和/或它们的DNA调控元件作为神经系统活动的示踪物的潜在用途，对于解剖参与处理和整合果蝇感觉信息的神经回路将是非常有价值的。此外，在冈比亚按蚊等使用化学线索识别寄主的病媒中，对进化上保守的基因的理解可能会导致新的昆虫控制策略。这些基因在引导寄主寻找行为的神经回路中发挥作用。

DESCRIPTION (provided by applicant): Genes that are transcribed in response to neuronal activity, named immediate early genes (IEGs), comprise members that are involved in important biological processes such as synaptic plasticity, learning and memory, and oncogenesis. A few genes have been identified as IEGs in mammals, but very little is known about IEGs in insects such as Drosophila melanogaster, which is used as a model to study these biological process. Furthermore, little is understood about genes that are downregulated by neuronal activity. We propose to undertake a genome-wide transcriptome analysis to identify genes that are both up-regulated and down- regulated in response to sensory neuron activity in three species of insects including Drosophila melanogaster, Drosophila pseudoobscura and the malaria vector Anopheles gambiae. By performing this analysis for three different species, we will be able to identify candidate IEGs for each species, as well as identify evolutionarily conserved IEGs that are regulated similarly across the three species. We propose to validate the identified candidate IEGs using a well-established quantitative RT-PCR.approach. Finally, we propose to map specificity of activity-induced transcription of IEGs to appropriate neurons using two alternative strategies, RNA in situ analysis and transgenic IEG-promoter analysis. For the transgenic strategy we propose to identify candidate regulatory regions of IEGs and test these promoter sequences in transgenic flies to determine whether they can drive reporter gene expression in response to neuronal activity. Successful completion of the proposed studies will provide a genome-wide catalog of genes that are regulated in an activity-dependent fashion in Drosophila and Anopheles. A successful method to transcriptionally report neuronal activity in vivo in insects would be a tremendous advance and could be applicable for the investigation of complex neurobiological problems including higher order processing of chemosensory information, chemsosensory coding, sensory integration and memory formation. PUBLIC HEALTH RELEVANCE: We propose to identify evolutionarily conserved genes that are regulated by activation of neurons in Drosophila and Anopheles using a genome-wide approach. In mammals activity-regulated genes have been associated with a number of important physiological processes such as nervous system function and cancer biology, and the ability to study them in the model insect Drosophila melanogaster will shed light on the fundamental mechanisms of their regulation and function. The potential use of these genes and/or their DNA regulatory elements as tracers of nervous system activity will be invaluable in dissecting neural circuits that are involved in processing and integrating sensory information in Drosophila. Furthermore, in disease vectors such as Anopheles gambiae that use chemical cues to identify their hosts, an understanding of evolutionarily conserved genes that function in neural circuits that guide host-seeking behaviors may lead to novel strategies for insect control.