Genome-wide Identification of Immediate Early Genes in Insects

描述（由申请人提供）：响应神经元活动而转录的基因，称为立即早期基因（IEG），包含参与重要生物过程的成员，例如突触可塑性、学习和记忆以及肿瘤发生。一些基因已被鉴定为哺乳动物中的 IEG，但对于昆虫（如果蝇）中的 IEG 知之甚少，而黑腹果蝇被用作研究这些生物过程的模型。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（包括黑腹果蝇、伪暗果蝇和疟疾媒介冈比亚按蚊）中响应感觉神经元活动而上调和下调的基因。通过对三个不同物种进行这种分析，我们将能够识别每个物种的候选 IEG，并识别在三个物种中受到类似调节的进化保守的 IEG。我们建议使用成熟的定量 RT-PCR 方法来验证已确定的候选 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）将活动诱导的 IEG 转录特异性映射到适当的神经元。对于转基因策略，我们建议鉴定 IEG 的候选调控区，并在转基因果蝇中测试这些启动子序列，以确定它们是否可以响应神经元活动驱动报告基因表达。拟议研究的成功完成将提供果蝇和按蚊中以活动依赖性方式调节的全基因组基因目录。一种成功地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，并且可适用于复杂的神经生物学问题的研究，包括化学感应信息的高级处理、化学感应编码、感觉整合和记忆形成。 公共健康相关性：我们建议使用全基因组方法来识别果蝇和按蚊神经元激活所调节的进化保守基因。在哺乳动物中，活性调节基因与许多重要的生理过程有关，例如神经系统功能和癌症生物学，在模型昆虫果蝇中研究它们的能力将揭示它们调节和功能的基本机制。这些基因和/或其 DNA 调控元件作为神经系统活动示踪剂的潜在用途对于解剖果蝇中参与处理和整合感觉信息的神经回路将具有无价的价值。此外，在冈比亚按蚊等使用化学线索识别宿主的疾病媒介中，了解在指导宿主寻找行为的神经回路中发挥作用的进化保守基因可能会带来新的昆虫控制策略。

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.