Isolation of cDNA encoding the constant region of the immuoglobulin heavy-chain from common carp (Cyprinus carpioL.)

Abstract

Complementary DNA fragments encoding the constant region (CH) of immunoglobulin heavy chain were isolated from spleen of carp (Cyprinus carpioL.) by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers corresponding to the conserved amino acid sequences in theJ-segment of the variable region, and in CH4. Sequences encoding CH4 and transmembrane domains were also obtained by a rapid amplification of cDNA end method (3′-RACE). Primary structure of the entire carp CH domain of the secreted form contained well conserved cysteine and tryptophan residues and showed 30% to 42% identity with that of other teleost species. Sequence analysis of membrane-bound CH domain revealed that the carp transmembrane domain is connected to the end of CH3, splicing out the entire CH4 exon, as in other teleosts. Interestingly, three distinct partial CH sequences which share 72–95% identity at amino acid level were obtained from a single carp. This strongly indicates the presence of duplicated CH genes, which are more divergent than those in Atlantic salmon, which carries two isotypic Ig H genes. Southern hybridisation using carp erythrocyte DNA also revealed two distinct CH genes indicating that each of the duplicated CH genes is disomicly inherited. It seems likely that the duplication in carp occurred as whole Ig H genes, in association with ancient tetraploidisation, rather than by tandem duplication, as reported for another tetraploidised teleost, Atlantic salmon.