IDENTIFICATION AND CHARACTERIZATION OF A NEW MAJOR HISTOCOMPATIBILITYCOMPLEX CLASS-I GENE IN CARP (CYPRINUS-CARPIO L)

In this study we report the finding of three representatives of a new group of major histocompatibility complex class I sequences in carp: C yca-12 (Cyca-UA101), a full-length cDNA; Cyca-SP1 (Cyca-UAW1), a poly merase chain reaction (PCR) fragment from cDNA; and Cyca-G11 (Cyca-UA1 02), a partial genomic clone. Comparison of the amino acid sequences of Cyca-12, Cyca-SP1, and Cyca-G11 with classical and non-classical cl ass I sequences from other species shows considerable conservation in regions that have been shown to be involved in maintaining the structu re and function of class I molecules. The genomic organization of Cyca -12 has been elucidated by analysis of a partial genomic clone Cyca-G1 1, in combination with PCR amplifications on genomic DNA of a homozygo us individual. Although the genomic organization is similar to that fo und in class I genes from other species, the 3' untranslated region co ntains an intron which is unprecedented in class I genes, and intron 2 is exceptionally large (+/-14 kilobases). Southern blot analysis indi cates the presence of multiple related sequences. In phylogenetic anal yses, the Cyca-UA sequences duster with class I genes from zebrafish a nd Atlantic salmon, indicating that the ancestral gene arose before th e salmonid/cyprinid split, approximately 120-150 million years ago. Th e previously reported class I Cyca-Z genes from carp and Caau-Z genes from goldfish cluster as a completely separate lineage. A polyclonal a ntiserum (anti-Cyca12) was raised against a recombinant fusion protein containing most of the extracellular domains of Cyca-12. The antibodi es showed substantial reactivity to the recombinant protein and an M(r ) 45 000 protein in membrane lysates of spleen and muscle, as well as to determinants present on leucocytes in fluorescence-activated cell s orter analyses. Erythrocytes and thrombocytes were found to be negativ e.