PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

跨学科的方法，涉及分子生物学和 电生理学，用于克隆和研究细胞的结构和表达 编码离子通道和神经递质受体的基因在 哺乳动物的大脑和骨骼肌和心肌被提出。主修 重点将放在开发一种新的通用方法来克隆这种 基因对那些通过蛋白质的传统方法的情况 提纯并不容易。该方法基于 细胞上表达的通道或受体的电生理学检测 向非洲爪哇卵母细胞显微注射mRNA的卵母细胞表面，联合 通过cdna克隆，以及杂交选择或杂交阻止程序。这个 首要目标将是分离编码电压敏感的基因 钙通道和5-羟色胺受体。人类的染色体基因 大脑、心脏和肌肉的电压敏感钠通道将 用标准方法从目前可用的部分cDNA克隆中分离 大脑基因。将特别强调确定 各通道基因产物多样性的分子基础 兴趣，以及这种多样性在信号处理中的意义。 在所有情况下，电生理的高精度表征 在卵母细胞中大量表达的通道的特性将是 采用单通道膜片钳方法。

An interdisciplinary approach, involving molecular biology and electrophysiology, for cloning and studying structure and expression of genes coding for ion channels and neurotransmitter receptors expressed in mammalian brain and skeletal and cardiac muscle is proposed. Major emphasis will be placed on developing a new general method for cloning such genes for those cases in which the conventional approach through protein purification is not readily applicable. The method is based on electrophysiological assays for channels or receptors expressed on the oocyte surface by microinjection of mRNA into Xenopus oocytes, combined with cDNA cloning, and hybrid selection or hybrid arrest procedures. The first goals will be the isolation of genes coding for voltage sensitive calcium channels and for serotonin receptors. The chromosomal genes for the voltage sensitive sodium channels of brain, heart, and muscle will be isolated by standard methods from a presently available partial cDNA clone for the brain gene. Special emphasis will be placed on determining the molecular basis of the diversity of gene products for all the channels of interest, and to the significance of this diversity in signal processing. In all cases, high precision characterization of the electrophysiological properties of the channels expressed in abundance in the oocyte will be carried out by single channel patch clamping methods.