GENOME RESPONSES TO DROSOPHILA ADDICTION USING DNA CHIPS

描述：（申请人的摘要）药物成瘾的机制似乎是 非常保守。因此，在动物模型系统中的工作可以极大地告知 我们对人类成瘾的理解规模经济和 果蝇的分子遗传学工具将被用来识别基因 和成瘾的遗传途径。最近对果蝇的研究 建立了可卡因和乙醇的行为敏感性模型。在 后者是健忘症的等位基因，健忘症是一种学习和记忆基因， 与刺激cAMP信号传导的脊椎动物神经肽同源， 鉴定拟南芥突变体显示出对乙醇的耐受性降低，这种缺陷 可以通过cAMP水平的药理学刺激来逆转。在前者中， 几个单基因突变体被证明会影响可卡因的致敏作用， 也由多巴胺能信号传导介导。这些研究证实了果蝇 作为可卡因和酒精成瘾的遗传模型系统， cAMP信号通路对两者的反应。 这个果蝇成瘾模型系统将随着DNA的使用而增强 微阵列芯片，以确定药物相关的基因表达的变化， 全基因组规模。在初步实验中，一个部分基因组原型 果蝇基因芯片已成功地从转录水平上鉴定出176个候选基因 在健忘症突变体中发生改变， cAMP水平的药理学操作。我们已经确认了一些 使用北方印迹分析或 定量PCR。从100多个芯片的初步分析，我们还 发展了一种新的通用统计方法来分析DNA芯片数据。 这些初步结果现在将与暴露于 可卡因和乙醇我们方法的核心是比较三个基因组， 反应：（1）药物暴露与对照，（2）药理学操作与 对照和（3）突变体与野生型。首先选择用于进一步体内研究 是那些在这三种情况下都有转录反应的基因 - 以及可卡因和乙醇的暴露。给定“函数的同调性” 已经在果蝇和脊椎动物的药物反应中观察到， 在果蝇中发现的基因很可能在脊椎动物中有同源物。因此这 这项工作将直接导致在成瘾的哺乳动物模型中的工作， 将使我们更好地了解人类成瘾的遗传基础。

DESCRIPTION: (Applicant's Abstract) Mechanisms of drug addiction appear to be remarkably conserved. Work in animal model systems can therefore greatly inform our understanding of human addiction. The economy-of-scale and molecular-genetic tools available in fruit flies will be used to identify genes and genetic pathways of addiction. Recent studies in Drosophila have established behavioral sensitivity models for both cocaine and ethanol. In the latter, an allele of amnesiac, a learning and memory gene that has some homology to a vertebrate neuropeptide that stimulates cAMP signaling, was identified. Amnesiac mutants show decreased tolerance to ethanol, a defect that can be reversed with pharmacological stimulation of cAMP levels. In the former, several single-gene mutants were shown to affect cocaine sensitization, which also was mediated by dopaminergic signaling. These studies validate Drosophila as a genetic model system for both cocaine and alcohol addiction and implicate the cAMP signaling pathway in the response to both. This Drosophila model system of addiction will be enhanced with the use of DNA microarray chips to identify drug-related changes in gene expression on a genomic-wide scale. In preliminary experiments, a partial genome prototype Drosophila chip has been used to identify 176 candidate genes transcriptionally altered in amnesiac mutants and 75 that are transcriptionally altered by pharmacological manipulation of cAMP levels. We have confirmed some of the statistical candidates from the chip using either Northern blot analysis or quantitative PCR. From initial analyses of more than 100 chips, we also have developed a novel and general statistical method to analyze DNA chip data. These initial results now will be integrated with data from exposures to cocaine and ethanol. Central to our approach is the comparison of three genomic responses: (1) drug-exposed vs. control, (2) pharmacological manipulation vs. control and (3) mutant vs. wild-type. Chosen first for further in vivo study will be those genes that are transcriptionally responsive in all three contexts - and to both cocaine and ethanol exposures. Given the "homology of function" already observed for drug responses in flies and vertebrates, the genes identified in flies most likely will have homologues in vertebrates. Hence this work will lead directly to work in mammalian models of addiction and ultimately will yield a greater understanding of the genetic basis of addiction in humans.