Related Experiment Videos
cDNA of the immunoglobulin kappa chain of an Epstein-Barr virus-transformed human lymphoid cell line: partial
Abstract:
We report the isolation, nucleotide sequence determination, and bacterial expression of a partial cDNA for the immunoglobulin kappa chain from the Epstein-Barr virus-transformed human lymphoid cell line GM131. The cDNA, cloned in pBR322 by use of oligo(dG) X oligo(dC) tails, yields two Pst I fragments of 250 and 600 base pairs (bp). Various restriction enzyme fragments of the cDNA were subcloned in the vectors M13 mp10 and M13 mp11 for sequence analysis. As a result of instability of the 250-bp M13 subclones, the base sequence of the 250-bp Pst I fragment could not be determined. The 600-bp Pst I fragment contains coding sequences for part of the variable (V) region (residues 78-95) and all of the joining (J) (residues 96-108) and constant (C) regions (residues 109-212) and extends 148 bp into the 3' flanking region. Although the C- and 3'-flanking-region sequences are identical to germ-line sequences, the J-region sequence does not correspond to any of the five human germ-line J regions. The sequence is most similar to that of J4, with three base changes resulting in one silent mutation and two amino acid substitutions, at residues 103 (Lys----Tyr) and 106 (Ile---Met). The silent mutation appears to be the result of RNA splicing between the J and the C regions. The V-region sequence differs from published V-region germ-line sequences at several codons and from the more common amino acid sequences at two positions, residues 91 and 93. At these positions, histidine residues are found in place of the more common tyrosine and serine, respectively. None of the four amino acid substitutions observed for the GM131 kappa-chain are unique, suggesting that the changes, which most likely contribute to antigenic specificity, are compatible with antibody structure and function. The 600-bp Pst I fragment was subcloned in two prokaryotic expression vectors, pATH11 and pUC8. In both instances, a kappa-chain fusion protein detectable by immunoblotting was produced.