A screen to identify mutations that disrupt alcohol tolerance in Drosophila

描述（由申请人提供）：尽管有大量不同的文献，但乙醇研究领域仍存在许多未解答的问题。近年来，关于乙醇对果蝇影响的研究有所扩大，但它仍然是一个年轻的领域，还有很多工作要做。使用果蝇作为乙醇模型系统的主要好处在于遗传操作的容易性和遗传修饰与行为表型相关联的速度。它是一个很好的“侦察生物”，可以识别候选基因，以便以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遗传筛选，以确定干扰果蝇对乙醇快速耐受性的突变。这是第一次在果蝇中进行乙醇筛选，专门寻找耐受性。由于测量的行为与以前使用的方法略有不同，我们可能会看到新的结果。我们采用两种方法。第一种方法是使用大的染色体缺失系统地筛选整个第三条染色体，然后将这些结果缩小到单个基因。第二种方法是筛选已经在其他模型系统中通过乙醇工作鉴定的候选基因的集合。使用这两种方法给了我们一个广泛的网络，以确定参与耐受性的基因。第二个具体目标是确定乙醇暴露后的时间基因表达模式。将从蝇头中纯化mRNA，并测定目的1中鉴定的基因的乙醇诱导的变化。为此，我将学习和使用实时RT-PCR。这将提供一个实质性的培训经验，这将有助于我的发展，作为一个全面的科学家。第三个目标是确定消除乙醇耐受性的突变是否也会影响乙醇偏好行为。将测试在目标1中鉴定的突变体，并且将使用不同的乙醇处理过程来观察基因型和环境对乙醇偏好的影响。苍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。完成这些目标不仅会立即增加乙醇研究领域（通过识别参与乙醇耐受性的新基因），还将增加对酒精中毒这一复杂疾病的遗传学的新见解。它将扩大使用果蝇作为乙醇模型系统的工具。通过研究果蝇的基因如何影响它们对酒精的反应，我们将了解酒精的影响，这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.