Genetic Analysis of specific expressions in mammalian brains.

从猫和大鼠脑皮质中提取RNA，并根据其核苷酸序列在体外酶促合成cDNA。可以获得小到大（0.5 - 6.5kb）的合成cDNA。将cDNA与<nu>从几个器官（肾、脾、肝和肺）中提取的polyA^+ mRNA（共50 μ g）杂交。通过羟基磷灰石层析（Bio Rad）从双链杂交体中分级分离单链cDNA。或者，用固定在膜滤器上的mRNA吸收cDNA。获得了少量吸收的cDNA。我们还获得<lambda>了猫和大鼠的gt 11 cDNA文库（两种动物文库均为2 - 3 x 10^6 μ g/ml）。用双向电泳和高压液相色谱法纯化了一种在牛脑中检测到的微量蛋白质（分子量68，000）。测定了该蛋白N端的16个氨基酸残基。用蛋白质的单克隆抗体筛选文库。本文还发展了一种非放射性标记DNA的方法，用于原位杂交实验，为下一步克隆脑特异性cDNA奠定了基础。用<lambda>1.3kb的琥珀酰琥珀酸裂解酶cDNA探针（Aval-Aval）从大鼠cDNA文库中筛选到一个阳性克隆。从噬菌体克隆中分离互补DNA并用限制性内切酶作图。大鼠脑cDNA的5 '端和3'端分别比肝cDNA的两端大800 bp和30 bp。有人认为，它们在结构上有共同的翻译目的。

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.