Fantoni A, Bozzoni I, Ullu E, Farace M G
Nucleic Acids Res. 1979 Aug 10;6(11):3505-17. doi: 10.1093/nar/6.11.3505.
Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.
从小鼠胎儿卵黄囊来源的红细胞中纯化出小鼠胚胎珠蛋白的信使核糖核酸。合成了与这些信使核糖核酸互补的双链脱氧核糖核酸，并将平端连接到经EcoRI酶切且用DNA聚合酶I修复的pBR322质粒上。在氨苄青霉素抗性转化体中，有一个含有携带珠蛋白特异性互补脱氧核糖核酸的质粒。插入序列约350个碱基对长。它含有一个EcoRI酶切位点和一个HinfI酶切位点，分别距离一端约170和80个碱基对。该插入片段不能被HindIII、HindII、BamHI、PstI、SalI、AvaI、TaqI、HpaII、BglI酶切。将编码α链以及x、y和z胚胎链的纯化信使核糖核酸混合物与重组质粒一起温育，然后在缺乏信使核糖核酸的网织红细胞裂解物蛋白质合成系统中对杂交后的信使核糖核酸进行翻译。通过羧甲基纤维素色谱法将翻译产物鉴定为z链。该重组质粒根据胚胎珠蛋白z被命名为“pBR322 - egz”。
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