CLONING OF EARLY RESPONSE GENES FROM THE NERVOUS SYSTEM

在化学或电刺激后，神经元依次表达和积累一系列早期反应基因(ERG)mRNAs，这些基因编码细胞因子、细胞表面受体、关键酶和转录因子(Tf)。这些基因调节和控制神经元对应激的反应。尽管它们很重要，但只有一小部分大脑特异的ERG被识别和克隆。为了响应这种用于神经系统基因发现的RFA，我们将应用一种新的亲和方法来选择性和直接分离大脑ERG mRNAs，基于它们的3‘非翻译区，AUUA不稳定重复。利用这项技术，我们已经从激活的淋巴细胞中制备了一个cDNA文库，其中包含各种已知的ERG cDNA以及大量可能编码未知ERG cDNA的未知cDNA。因此，我们建议从静息和海人藻酸处理的小鼠脑和海马区中分离出mRNA。构建AUUUA筛选、ERG基因文库；对文库中的所有克隆进行部分测序，并鉴定独特的cDNA；3)分离编码新ERG的全长cDNA和4)。用原位杂交法研究新的ERGs在小鼠脑内的表达模式。总而言之，这些研究将识别迄今未知的由癫痫诱发的ERG，并开始表征这些基因在大脑中的时空表达。

After chemical or electrical excitation, neurons sequentially express and accumulate a battery of early response gene (ERG) mRNAs which code for cytokines, cell surface receptors, critical enzymes and transcription factors (TF). These genes mediate and control neuronal responses to stress. Despite their importance, only a small number of brain specific ERGs have been identified and cloned. In response to this RFA for nervous system gene discovery, we will apply a novel affinity method for the selective and direct isolation of brain ERG mRNAs based upon their 3' untranslated region, AUUUA instability repeats. Using this technology, we have prepared a cDNA library from activated lymphocytes which contain a variety of known ERG cDNAs as well as a substantial number of unknown cDNAs which potentially code for uncharacterized ERG cDNAs. Therefore, we propose to isolate mRNA from resting and kainic acid treated mouse brain and hippocampus and 1). Produce AUUUA selected, ERG cDNA libraries; 2). Partially sequence all clones in the libraries and identify unique cDNAs; 3). Isolate full length cDNAs coding for novel ERGs and 4). Characterize the expression patterns of novel ERGs in mouse brain by in situ hybridization. In aggregate, these studies will identify heretofore unknown, ERGs induced by seizure and begin to characterize the temporal and spatial expression of these genes in the brain.