Genetic Analysis of specific expressions in mammalian brains.

从猫和大鼠的大脑皮层中提取RNA，根据其核苷酸序列，在体外酶促合成cDNAs。可以获得小到大(0.5-6.5kb)的合成的cDNA。将从肾、脾、肝、肺等器官中提取的Polya^+mRNAs(共50个)与cDNA杂交。用Bio-Rad从双链杂交体中分离出单链cDNA。或者，将cDNA与固定在膜过滤器上的mRNAs一起吸附。获得了少量的被吸附的DNA。我们还获得了猫和大鼠的&lt；lambda&gt；gt；11c DNA文库(两个文库均为2-3×10^6噬菌体/毫升)。从牛脑中检测到一种相对分子质量为68,000的蛋白质，用双向凝胶电泳法和高压液相色谱分离纯化。测定了该蛋白N端的16个氨基酸残基。文库已经用抗该蛋白的单抗进行了筛选。还发展了一种非放射性标记DNA的替代方法，用于原位杂交实验，该方法应针对以下脑特异性cDNA的克隆。我们可以得到很多关于它的信息。用精氨酸琥珀酸裂解酶基因探针(1.3kb；Aval-Aval)从表达载体&lt；lambda&gt；gt 11文库中平行获得阳性克隆。从噬菌体克隆中提取互补DNA，并用限制性内切酶进行定位。大鼠脑的5‘端和3’端分别比大鼠肝的5‘端和3’端大800bp和30bp。这表明它们在结构上具有共同的翻译表达。

RNAs were extracted from brain cortexes of cats and rats, and cDNA were synthesized enzymatically in vitro on the basis of their nucleotide sequences. Small to large sizes (0.5 - 6.5 kb) of the synthesized cDNAs could be obtained. The cDNAs were hybridized with polyA^+mRNAs (total 50<nu>g) extracted from several organs (kidney,spleen,liver and lung). Single stranded cDNAs were fractionated from double stranded hybrids by hydroxyappatite chromatography (Bio Rad). Alternatively,the cDNAs were absorbed with the mRNAs immobilized on membrane filters. Small amounts of the absorbed cDNAs were obtained. We also obtained <lambda>gt 11 cDNA libralies of cat and rat(2 - 3 x 10^6 phages/ml for both animal libralies). A protein (m.w.68,000) which was detected with small amounts in cow brains was purified by two-dimentional electrophoresis and high pressure liquid chromatography. The 16 amino acid residues at N-end of the protein were determined. The libralies have been screened with monoclonal antibodies to the protein. An alternative non-radioactive method for labeling DNA was also developed for the in situ hybridization experiments,which should be aimed at the followings of the cloning for brain specific cDNAs. We could get much informations of it. A positive clone was paralelly obtained from the expression vector <lambda>gt 11 libralies with rat cDNAs, using a cDNA probe (1.3 kb; Aval-Aval) of argininosuccinate lyase. Complementary DNA was isolated from the phage clone and mapped with restriction enzymes. The rat brain cDNA was 800bp and 30 bp larger at the 5'and3'ends,respectively,in comparison with each end of the rat liver cDNA. It was suggested that they had structurally common translated resion.