Sousa R, Tannery N H, Zhou S, Lafer E M
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
J Neurosci. 1992 Jun;12(6):2130-43. doi: 10.1523/JNEUROSCI.12-06-02130.1992.
A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse-specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1-20. Immunohistochemical and biochemical analyses revealed that the F1-20 protein is synapse associated. Western blot analyses revealed that the F1-20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1-20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1-20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1-20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1-20 protein by immunohistochemistry, and of the F1-20 mRNA by in situ hybridization. We found that the F1-20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1-20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1-20 protein are found in the cortically organized regions of the brain. The highest levels of F1-20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1-20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1-20 mRNA between different nuclear groups.
要全面理解突触功能，需要对神经末梢进行分子层面的描述。本研究的目标是通过鉴定一种新型的突触特异性蛋白，为这种描述做出贡献。我们筛选了一个单克隆抗体文库，寻找针对突触蛋白的抗体。然后利用这些抗体通过表达筛选来分离cDNA克隆。在此，我们报告了与单克隆抗体F1 - 20反应的蛋白的详细特征。免疫组织化学和生化分析表明，F1 - 20蛋白与突触相关。蛋白质印迹分析显示，F1 - 20蛋白是一种脑特异性多肽，在SDS - PAGE上的表观分子量为190,000道尔顿。Northern印迹分析表明，从F1 - 20 cDNA克隆产生的探针与一个约4.8千碱基的单一脑特异性mRNA杂交。原位杂交实验表明，F1 - 20 mRNA的表达具有神经元特异性。Northern和蛋白质印迹分析表明，F1 - 20 mRNA水平在出生后第4天急剧增加，蛋白质水平在出生后第7天急剧增加。这与小鼠中枢神经系统中活跃的突触发生和突触成熟时期相对应。我们通过免疫组织化学对F1 - 20蛋白的神经解剖分布进行了表征，并通过原位杂交对F1 - 20 mRNA进行了表征。我们发现，F1 - 20 mRNA和蛋白在脑中的表达并不均匀。F1 - 20蛋白表达的变化很复杂，并且揭示了针对其他突触相关分子的探针也呈现出的模式。F1 - 20蛋白水平最高的区域位于大脑的皮质组织区域。F1 - 20 mRNA水平最高的区域位于长距离投射神经元中。不同类型的大输出神经元之间，F1 - 20 mRNA的表达也存在差异，不同核团之间F1 - 20 mRNA的表达也有广泛差异。
