A screen to identify mutations that disrupt alcohol tolerance in Drosophila

描述(由申请人提供)：尽管有广泛而多样的文献，但乙醇研究领域仍有许多悬而未决的问题。近年来，关于乙醇对黑腹果蝇影响的研究有所扩大，但这仍然是一个年轻的领域，有很多工作要做。使用果蝇作为酒精模型系统的主要好处在于遗传操作的简便性和遗传修改与行为表型联系的速度。它是一个很好的“侦察有机体”，可以识别候选基因，然后在更复杂的系统中进行测试。在第一个具体目标中，我们将进行大规模的基因筛查，以确定扰乱果蝇对乙醇快速耐受性的突变。这是果蝇中第一个专门寻找耐受性的乙醇筛查。由于测量的行为与以前使用的方法略有不同，我们很可能会看到新的结果。我们正在使用两种方法。第一种方法是使用大的染色体缺失来系统地筛选整个第三条染色体，然后将结果缩小到单个基因。第二种方法是筛选一组候选基因，这些基因已经被乙醇在其他模型系统中识别出来。使用这两种方法给我们提供了一个广泛的网络来识别与耐受有关的基因。第二个具体目标是确定酒精暴露后的时间基因表达模式。将从苍蝇头中提纯mRNA，并将检测Aim 1中确定的基因是否有乙醇诱导的变化。为此，我将学习并使用实时RT-PCR。这将提供大量的培训经验，有助于我成为一名全面发展的科学家。第三个目标是确定消除酒精耐受性的突变是否也会影响酒精偏好行为。将对目标1中确定的突变体进行测试，并将使用不同的乙醇处理过程来观察基因和环境对乙醇偏好的影响。果蝇对乙醇有天然的偏好，因为它存在于腐烂的水果中，所以它们是进行这些测试的理想模型系统。完成这些目标不仅将立即增加乙醇研究领域(通过识别与酒精耐受性有关的新基因)，还将为酒精中毒这一复杂疾病的遗传学提供新的见解。它将扩大现有的工具，将果蝇用作乙醇模型系统。通过研究果蝇的基因如何影响它们对酒精的反应，我们将对酒精的影响有一个了解，这将有助于未来治疗和预防酒精中毒。

DESCRIPTION (provided by applicant): Despite a wide and varied body of literature, many unanswered questions remain in the field of ethanol research. Research into ethanol's effects with Drosophila melanogaster has expanded in recent years, but it still remains a young field with much to be done. The key benefit in using Drosophila as a model system for ethanol lies in the ease of genetic manipulation and the speed at which genetic modifications can be linked to behavioral phenotypes. It is a good "scouting organism" to identify candidate genes that can later be tested in more complex systems. In the first specific aim, we will conduct a large scale genetic screen to identify mutations that perturb rapid tolerance to ethanol in Drosophila. This is the first ethanol screen in Drosophila to look specifically for tolerance. Since the behavior being measured is slightly different than methods previously used, we are likely to see new results. We are using two approaches. The first is to systematically screen the entire third chromosome using large chromosomal deletions, then narrow down those results to single genes. The second approach is to screen a collection of candidate genes that have been identified by ethanol work in other model systems. Using both approaches gives us a wide net to identify genes involved in tolerance. The second specific aim is to determine the temporal gene expression patterns following ethanol exposure. mRNA will be purified from fly heads and the genes identified in aim 1 will be assayed for ethanol induced changes. For this aim, I will learn and use real-time RT-PCR. This will provide a substantial training experience that will aid in my development as a well rounded scientist. The third aim is to determine whether mutations that eliminate ethanol tolerance also influence ethanol preference behavior. The mutants identified in aim 1 will be tested, and various courses of ethanol treatment will be used to see the effects of both genotype and environment on preference to ethanol. Flies have a natural preference for ethanol, due to its presence in rotting fruit, so they are an ideal model system with which to conduct these tests. Completing these aims will not only add immediately to the ethanol research field (by identifying new genes involved in ethanol tolerance), it will add new insight into the genetics of the complex disease of alcoholism. It will expand the tools in place for the use of Drosophila as an ethanol model system. By studying how the genes of fruit flies affect their response to alcohol, we will gain an understanding of alcohol's effects that helps treatment and prevention of alcoholism in the future.