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）mRNA，其编码细胞因子、细胞表面受体、关键酶和转录因子（TF）。 这些基因介导和控制神经元对压力的反应。 尽管它们的重要性，只有少数脑特异性ERG已被确定和克隆。 为了响应神经系统基因发现的RFA，我们将应用一种新的亲和方法，基于其3'非翻译区AUUUA不稳定重复序列选择性和直接分离脑ERG mRNA。 使用这种技术，我们已经从活化的淋巴细胞中制备了一个cDNA文库，该文库含有多种已知的ERG cDNA以及大量可能编码未表征的ERG cDNA的未知cDNA。 因此，我们建议从静息和红藻氨酸处理的小鼠脑和海马中分离mRNA，和1）。产生AUUUA选择的ERG cDNA文库; 2）.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）.通过原位杂交表征小鼠脑中新型ERG的表达模式。 总之，这些研究将确定迄今为止未知的，由癫痫发作诱导的ERG，并开始表征这些基因在大脑中的时间和空间表达。

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.