DIFFERENTIAL PCR SCREEN FOR LONG TERM MEMORY

长期记忆（LTM）形成的普遍保守性质是 需要在cAMP的控制下进行级联的基因表达 反应性增强子结合蛋白（CREB）。 然而，在任何情况下， 激活的LTM基因在一个系统中被鉴定， 遗传分析 本研究的主要目的是 分离和鉴定果蝇中CREB靶基因，并开始 分析他们在LTM中的角色。 丰富的分子和基因序列 果蝇中可用的方法，以及 建立了长期联想记忆的标准测定法，使 果蝇是这个项目的一个有吸引力的系统。 CREB靶基因将使用称为 差异mRNA显示。 这是一种基于PCR的技术， 成功地用于分离在两个细胞中表达不同的mRNA， 感兴趣的组织。 在这种情况下，差异显示将被应用于 从两组不同果蝇的脑组织中分离的mRNA。 在 第一组，将诱导编码CREB阻断剂同种型的转基因， 一个被证明可以专门阻止LTM的程序（见附录）。 在第二组中，编码CREB激活物同种型的转基因将被转染。 诱导。 第二种方法已被证明可以增强LTM的形成。 在该筛选中分离的PCR片段将用于RNA酶保护 和原位杂交分析，以确认CREB/LTM响应 调控 然后将候选片段用于筛选大脑cDNA 使用标准技术的库。 新cDNA在LTM中的作用 然后通过标准的错误表达和诱变来测试形成 接近。

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.