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Heavy Chain Diversity Region Segments of the Channel Catfish: Structure, Organization, Expression and Phylogenetic Implications^{1 ,2}

Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216

Circular DNA, derived from lymphocytes of juvenile channel catfish, was used to construct libraries that were screened to identify the products of immunoglobulin D_H-J_H excision events. Clones were characterized that contained D_H to J_H recombination signal joints. The signal joints represented 23-bp recombination signal sequences (RSS) identical to germline J_H segments that were adjacent to D_H 12-bp RSS elements. D_H flanking regions within the clones were used to probe a genomic library. Three germline D_H gene segments containing 11–19 bp coding regions flanked by 12-bp RSS elements with conserved heptamers and nonamers were identified. The DH locus is closely linked to the J_H locus, and Southern blots indicate that the D_H segments represent different single member gene families. Analysis of H chain cDNA shows that each germline D_H segment was expressed in functional VDJ recombination events involving different J_H segments and members of different V_H families. Several aspects of CDR3 junctional diversity were evident, including deletion of coding region nucleotides, N- and P-region nucleotide additions, alternate D_H reading frame utilization, and point mutations. Coding region motifs of catfish D_H segments are phylogenetically conserved in some D_H segments of higher vertebrates. These studies indicate that the structure, genomic organization, and recombination patterns of D_H segments typically associated with higher vertebrates evolved early in vertebrate phylogeny at the level of the bony fish.