Kumanishi T, Usui H, Ichikawa T, Nishiyama A, Katagiri T, Abe S, Yoshida Y, Washiyama K, Kuwano R, Sakimura K
Department of Neuropathology, Niigata University, Japan.
Acta Neuropathol. 1992;83(6):569-78. doi: 10.1007/BF00299404.
We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.
我们从胶质瘤细胞系U - 251 MG中分离出三个胶质纤维酸性蛋白（GFAP）cDNA克隆。从U - 251 MG cDNA文库中分离出的一个克隆较长，但两端缺失。利用poly(A)+RNA和根据该克隆序列合成的引物，我们采用聚合酶链反应辅助的cDNA末端快速扩增（PCR - RACE）方法（一种分离cDNA末端的策略），获得了5'和3'末端的cDNA克隆。根据这些重叠克隆的序列，确定了人GFAP cDNA的完整核苷酸序列。起始（ATG）和终止（TGA）信号分别位于核苷酸位置15和1311，将3027 bp的整个序列分为5'非编码区的14 bp、编码区的1296 bp和3'非编码区的1717 bp。使用由编码区和3'非编码区制备的cDNA探针，对来自人和啮齿动物大脑以及人GFAP阳性和阴性细胞的RNA进行了两种不同严谨度的Northern印迹杂交。结果表明，3'非编码区探针比编码区探针对人GFAP更具特异性，编码区探针仅在较高严谨度条件下具有特异性。各种中间丝蛋白序列的同源性分析也表明了这一点。基于这些发现，我们对分选的人染色体进行了斑点印迹杂交，并对一组仓鼠 - 人杂种体细胞的PCR扩增DNA进行了Southern印迹杂交，将人GFAP基因定位到了17号染色体上。
