CHARACTERIZATION OF HUMAN CDNA AND GENOMIC CLONES FOR GLIAL FIBRILLARY ACIDIC PROTEIN
CHARACTERIZATION OF HUMAN CDNA AND GENOMIC CLONES FOR GLIAL FIBRILLARY ACIDIC PROTEIN
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DOI：
10.1016/0169-328x(90)90078-r
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发表时间：
1990-05-01
期刊：
影响因子：
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通讯作者：
FREESE, E
中科院分区：
文献类型：
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作者：
BRENNER, M;LAMPEL, K;FREESE, E
已分离出人gfa的部分cDNA克隆和完整的基因组克隆，gfa是编码星形胶质细胞中间丝的主要成分胶质细胞酸性蛋白（GFAP）的基因。测定了整个编码区和5“侧翼DNA的102 bp的核苷酸序列。通过引物延伸和探针保护实验确定mRNA起始位点，然后使用一种新的体外转录和翻译程序来建立mRNA中的第一个ATG启动GFAP合成。预测的人GFAP的氨基末端序列与先前从其基因序列推断的小鼠GFAP的氨基末端序列有很大不同，尽管在其他方面具有很高的同源性。通过确定已发表的小鼠基因组序列具有不正确的额外碱基来解决这种差异。校正的序列在其预测的氨基酸序列中产生人和小鼠GFAP之间的强同源性，并且表明人和小鼠GFAP起始于同源位置。这里推导出的两种蛋白质的起始序列与先前通过直接测序其氨基末端获得的猪GFAP的起始序列密切匹配。这支持了所提出的蛋白质起始位点，也表明GFAP在其氨基末端不被加工。
Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). The nucleotide sequence of the entire coding region and 102 bp of the 5'' flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to establish that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resoved by determining that the published mouse genomic sequence has an incorrect additional base. The corected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.