During the analysis of Ig superfamily members within the available rainbow trout (Oncorhynchus mykiss) EST gene index, we identified a unique Ig heavy-chain (IgH) isotype. cDNAs encoding this isotype are composed of a typical IgH leader sequence and a VDJ rearranged segment followed by four Ig superfamily C-1 domains represented as either membrane-bound or secretory versions. Because teleost fish were previously thought to encode and express only two IgH isotypes (IgM and IgD) for their humoral immune repertoire, we isolated all three cDNA isotypes from a single homozygous trout (OSU-142) to confirm that all three are indeed independent isotypes. Bioinformatic and phylogenetic analysis indicates that this previously undescribed divergent isotype is restricted to bony fish, thus we have named this isotype ''IgT'' () for teleost fish. Genomic sequence analysis of an OSU-142 bacterial artificial chromosome (BAC) clone positive for all three IgH isotypes revealed that IgT utilizes the standard rainbow trout VH families, but surprisingly, the IgT isotype possesses its own exclusive set of DH and JH elements for the generation of diversity. The IgT D and J segments and constant (C) region genes are located upstream of the D and J elements for IgM, representing a genomic IgH architecture that has not been observed in any other vertebrate class. All three isotypes are primarily expressed in the spleen and pronephros (bone marrow equivalent), and ontogenically, expression of IgT is present 4 d before hatching in developing embryos.development ͉ immunoglobulin A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells through the process of V(D)J recombination. This process is restricted solely to gnathostomes (jawed vertebrates); no evidence for either antibody gene fragments or the VDJrecombinase machinery has been identified in agnathan fish (lampreys and hagfish) (1, 2). The specific effector function (complement fixation, recognition by phagocytic cells, and secretion in mucosal tissues) depends on the nature of the isotype constant (C) region. In mammals, there are five Ig isotypes that possess distinct effector functions for secretory immunity. Ig⌴ is the only antibody isotype found universally in gnathostomes, and until 1997, teleosts (bony fish) were thought to possess only Ig⌴; however, research on catfish (3) and, later, on Atlantic salmon (4) provided evidence for the existence of IgD in teleosts. The genomic location of the teleost delta gene (␦) immediately downstream of , coupled with modest sequence identity to mammalian ␦ and coexpression of IgM and IgD in catfish B cell lines, solidified the relationship of teleost IgD to that of mammals. Expression of teleost IgD, like that of mammals, is achieved by alternative splicing from the rearranged VDJ gene to the C ␦ genes, with the exception that the teleost IgD message retains the first exon of C 1, thus forming a chimeric antibody isotype. After the discovery of Ig heavy-chain (IgH) genes in elasmob...

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