ISOLATION OF CDNA-ENCODING THE CONSTANT-REGION OF THE IMMUNOGLOBULIN HEAVY-CHAIN FROM COMMON CARP (CYPRINUS-CARPIO L.)

Complementary DNA fragments encoding the constant region (CH) of immun oglobulin heavy chain were isolated from spleen of carp (Cyprinus carp io L.) by reverse transcription-polymerase chain reaction (RT-PCR) usi ng degenerated primers corresponding to the conserved amino acid seque nces in the J-segment of the variable region, and in CH4. Sequences en coding CH4 and transmembrane domains were also obtained by a rapid amp lification of cDNA end method (3'-RACE). Primary structure of the enti re carp CH domain of the secreted form contained well conserved cystei ne 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. Interest ingly, three distinct partial CH sequences which share 72-95% identity at amino acid level were obtained from a single carp. This strongly i ndicates 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 d istinct CH genes indicating that each of the duplicated CH genes is di somicly inherited. It seems likely that the duplication in carp occurr ed as whole Ig H genes, in association with ancient tetraploidisation, rather than by tandem duplication, as reported for another tetraploid ised teleost, Atlantic salmon. (C) 1998 Academic Press.