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，称为即时早期基因（IEGs），包括参与重要生物过程的成员，如突触可塑性，学习和记忆以及肿瘤发生。一些基因在哺乳动物中被鉴定为eeg，但对昆虫（如黑腹果蝇）的eeg知之甚少，后者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，以确定三种昆虫（包括黑腹果蝇、伪腹果蝇和疟疾媒介冈比亚按蚊）的感觉神经元活动响应中上调和下调的基因。通过对三个不同的物种进行分析，我们将能够确定每个物种的候选eggs，以及确定进化上保守的eggs，这些eggs在三个物种中受到相似的调节。我们建议使用成熟的定量rt - pcr方法验证已确定的候选eeg。最后，我们建议使用RNA原位分析和转基因eeg启动子分析两种替代策略，将活动诱导的eeg转录特异性映射到适当的神经元上。对于转基因策略，我们建议确定候选的eeg调控区域，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驱动报告基因表达以响应神经元活动。成功完成拟议的研究将提供果蝇和按蚊以活动依赖的方式调节的基因全基因组目录。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，可用于研究复杂的神经生物学问题，包括化学感觉信息的高阶处理、化学感觉编码、感觉整合和记忆形成。

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.