Characterization and molecular cloning of the mRNA for the heavy (epsilon) chain of rat immunoglobulin E.

Publisher Website
Google Scholar
Abstract
The mRNA for the H (.epsilon.) chain of rat IgE was studied. Cytoplasmic RNA was prepared from the 2 rat immunocytomas IR2 and IR162 and fractionated by sucrose gradient centrifugation. An enriched fraction containing .apprxeq. 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 .apprxeq. 2200 nucleotides long and constitutes a minute fraction in the total mRNA both in the IR2 and the IR162 tumors, unlike the mRNA for L chains. Double-stranded c[complementary]DNA copies prepared from 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, 1 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. A sequence is reported 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.