Structure, genomic organization, and phylogenetic implications of six new VH families in the channel catfish.

To define members of previously unknown VH gene families, a channel catfish immunoglobulin heavy chain cDNA library was constructed and screened with probes specific for the seven known catfish VH families. Reiterative screening and sequence studies defined six new VH families, designated VH8-VH13, which brings the total number of VH families in the catfish to 13. This is the highest number of VH families presently defined in a lower vertebrate. Sequence comparisons indicate there is extensive diversity between members of different families with the greatest variability encoded within the complementarity determining regions. Genomic libraries were screened, and germline VH segments representing each of these new families were identified. The VH segments are closely linked and interspersed with members of different VH families. Each of these germline gene segments shared characteristic structural features: an upstream region that contained transcriptional regulatory elements, a leader sequence split by a short intron, an open reading frame encoding readily identified framework and complementarity determining regions, and a terminal recombination signal sequence consisting of a consensus heptamer, a 22-24 bp spacer with conserved 5'- and 3'-ends, and a consensus A-rich nonamer. Southern blot analyses estimate the number of members within these new families ranges from small (2-7 members in VH9, VH10, and VH12) to medium (9-13 members in VH8, VH11, and VH13). Thus, there are between 165 and 200 germline VH segments represented by these combined 13 families with present analyses indicating that perhaps one-half of these are pseudogenes. Phylogenetic comparisons indicate that members of these different catfish VH families cluster within Groups C and D of vertebrate VH genes. These analyses also indicate that Group D is represented by two different branches and both branches include VH families from different lineages of bony fish that diverged early in vertebrate phylogeny.