DIFFERENTIAL PCR SCREEN FOR LONG TERM MEMORY

长时记忆(LTM)形成的一个普遍保守的性质是 CAMP控制下基因表达级联的要求 反应性增强子结合蛋白(CREB)。然而，在任何情况下，CREB都没有 激活的LTM基因已在一种服从于 基因分析。这里提出的研究的主要目标是 分离和鉴定果蝇CREB靶基因，并开始 分析它们在LTM中的作用。丰富的分子和基因序列 在果蝇中可用的方法，以及S的可用性 建立了长期联想记忆的标准检测方法 果蝇是这个项目的一个有吸引力的系统. CREB靶基因将使用一种名为 差异显示的mRNA。这是一种基于聚合酶链式反应的技术 成功地用于分离在两个基因中表达不同的mRNA 感兴趣的组织。在这种情况下，差异显示将应用于 从两组不同果蝇的脑组织中提取的mRNA.在 第一组，将诱导编码CREB阻滞剂亚型的转基因， 如图所示，该程序专门阻止LTM(见附录)。 在第二组中，编码CREB激活剂亚型的转基因将是 诱导性。这第二个过程已经被证明可以促进LTM的形成。 在此筛选中分离的PCR片段将用于RNA酶保护 和原位杂交分析以确认CREB/LTM反应 监管。然后，候选片段将被用于筛选大脑cDNA 使用标准技术的库。新的cDNA在LTM中的作用 然后将通过标准的缺失表达和突变来测试形成 接近了。

A universally conserved property of long-term memory (LTM) formation, is the requirement for a cascade of gene expression under control of the cAMP responsive enhancer binding protein (CREB). However in no case have CREB activated LTM genes been identified in a system which is amenable to genetic analysis. Thr primary goal of the research proposed here is to isolate and characterize CREB target genes in Drosophila, and begin an analysis of their role in LTM. The rich array of molecular and genetic approaches available in Drosophila, as well s the availability of established standard assays for long-term associative memory, make Drosophila an attractive system for this project. CREB target genes will be identified using a technique known as differential mRNA display. This is a PCR based technique which as been successfully used to isolate mRNAs that are expressed differently in two tissues of interest. In this case, differential display will be applied to mRNA isolated from brain tissue from two different groups of flies. In the first group, a transgene encoding a CREB blocker isoform will be induced, a procedure which as been shown to specifically block LTM (see appendix). In the second group, a transgene encoding a CREB activator isoform will be induced. This second procedure has been shown to enhance LTM formation. PCR fragments isolated in this screen will be used for RNAase protection and insitu hybridization analyses to confirm CREB/LTM responsive regulation. Candidate fragments will then be used to screen brain CDNA libraries using standard techniques. The role of novel cDNAs in LTM formation will then be tested by standard miss-expression and mutagenesis approaches.