Ohkubo H, Kageyama R, Ujihara M, Hirose T, Inayama S, Nakanishi S
Proc Natl Acad Sci U S A. 1983 Apr;80(8):2196-200. doi: 10.1073/pnas.80.8.2196.
A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxyl-terminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRag16, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecular consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.
合成了与指定血管紧张素羧基末端序列（异亮氨酸 - 组氨酸 - 脯氨酸 - 苯丙氨酸 - 组氨酸）的密码子互补的十四聚体寡聚脱氧核糖核苷酸混合物，将其作为两个库使用，用于从大鼠肝脏mRNA序列的cDNA文库中分离血管紧张素原特异性的cDNA克隆。这两个库（寡核苷酸1和寡核苷酸2），每个库包含24个寡聚脱氧核糖核苷酸，首先用作引物引发大鼠肝脏mRNA的逆转录。发现其中一个库（寡核苷酸1）引发了一个约160个核苷酸的特异性32P标记的cDNA，该cDNA包含与大鼠血管紧张素氨基酸序列完全对应的反编码序列。反过来，这个cDNA被用于重新筛选通过最初用寡核苷酸1混合物杂交选择大鼠肝脏cDNA文库而分离出的cDNA克隆。由此获得的一个克隆，命名为pRag16，进行了核苷酸序列分析，并证实其包含编码大鼠血管紧张素原前体的近乎全长的cDNA序列。推导的氨基酸序列表明前体分子由453个氨基酸残基的血管紧张素原和24个氨基酸残基的假定信号肽组成。血管紧张素原的预测分子量和氨基酸组成与使用纯化蛋白测定的结果非常吻合。血管紧张素部分位于血管紧张素原的氨基末端部分，直接位于信号肽之前，后面跟着一个大的羧基末端序列，该序列包含两个内部同源序列和三个潜在的糖基化位点。
