Identification and characterization of a new major histocompatibility complex class I gene in carp (Cyprinus carpio...

In this study we report the finding of three representatives of a new group of major histocompatibility complex class I sequences in carp: Cyca-12 (Cyca-UA1*01), a full-length cDNA; Cyca-SP1 (Cyca-UAW1), a polymerase 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 class I sequences from other species shows considerable conservation in regions that have been shown to be involved in maintaining the structure and function of class I molecules. The genomic organization of Cyca-12 has been elucidated by analysis of a partial genomic clone (Cyca-G11, in combination with PCR amplifications on genomic DNA of a homozygous individual. Although the genomic organization is similar to that found in class I genes from other species, the 3' untranslated region contains an intron which is unprecedented in class I genes, and intron 2 is exceptionally large (+/-14 kilobases). Southern blot analysis indicates the presence of multiple related sequences. In phylogenetic analyses, the Cyca-UA sequences cluster with class I genes from zebrafish and Atlantic salmon, indicating that the ancestral gene arose before the salmonid/cyprinid split, approximately 120-150 million years ago. The previously reported class I Cyca-Z genes from carp and Caau-Z genes from goldfish cluster as a completely separate lineage. A polyclonal antiserum (anti-Cyca12) was raised against a recombinant fusion protein containing most of the extracellular domains of Cyca-12. The antibodies showed substantial reactivity to the recombinant protein and an Mr 45000 protein in membrane lysates of spleen and muscle, as well as to determinants present on leucocytes in fluorescence-activated cell sorter analyses. Erythrocytes and thrombocytes were found to be negative.