Hellman L, Pettersson U, Bennich H
Proc Natl Acad Sci U S A. 1982 Feb;79(4):1264-8. doi: 10.1073/pnas.79.4.1264.
We report a study of the mRNA for the heavy (epsilon) chain of rat IgE. Cytoplasmic RNA was prepared from the two rat immunocytomas IR2 and IR162 and fractionated by sucrose gradient centrifugation. An enriched fraction containing approximately 5% mRNA for the epsilon chain was obtained in this way. When translated in vitro, it produced a 59,000-dalton polypeptide, which in the presence of a membrane fraction yielded a 90,000-dalton polypeptide, presumably through posttranslational modification. Both polypeptides were precipitated by rabbit antisera that were monospecific for rat epsilon chains. The epsilon chain mRNA was estimated to be approximately 2200 nucleotides long and constitutes a minute fraction in the total mRNA both in the IR2 and the IR162 tumors, unlike the mRNA for light chains. Double-stranded cDNA copies prepared frm the RNA fraction, which was enriched for epsilon chain mRNA, were inserted into the Pst I cleavage site of the pBR322 vector. Twenty clones with inserts exceeding 1000 base pairs were used for selection of mRNA from the IR2 tumor. By in vitro translation of the selected mRNA, one clone was identified that yielded a polypeptide with the same size as the unprocessed epsilon chain. The nucleotide sequence was determined for part of the inserted cDNA in this candidate clone and was found to be homologous to a sequence in the constant region (C) of the human epsilon chain. In this communication we report a sequence from the C epsilon 3 domain of the rat IgE. When compared to the corresponding sequence of human IgE, 55% of the amino acids in the rat sequence were found to be conserved.
我们报告了一项关于大鼠IgE重链（ε链）mRNA的研究。从两种大鼠免疫细胞瘤IR2和IR162中制备细胞质RNA，并通过蔗糖梯度离心进行分级分离。通过这种方法获得了一个富含约5%ε链mRNA的级分。当在体外进行翻译时，它产生了一个59,000道尔顿的多肽，在膜组分存在的情况下，可能通过翻译后修饰产生了一个90,000道尔顿的多肽。两种多肽都能被对大鼠ε链具有单特异性的兔抗血清沉淀。据估计，ε链mRNA约有2200个核苷酸长，在IR2和IR162肿瘤的总mRNA中只占极小的一部分，这与轻链mRNA不同。从富含ε链mRNA的RNA级分制备的双链cDNA拷贝被插入到pBR322载体的Pst I切割位点。使用20个插入片段超过1000个碱基对的克隆从IR2肿瘤中筛选mRNA。通过对所选mRNA进行体外翻译，鉴定出一个克隆，它产生的多肽与未加工的ε链大小相同。测定了该候选克隆中插入的cDNA部分的核苷酸序列，发现它与人类ε链恒定区（C）的一个序列同源。在本通讯中，我们报告了大鼠IgE的Cε3结构域的一个序列。与人类IgE的相应序列相比，发现大鼠序列中55%的氨基酸是保守的。
