THE GENERATION OF ANTIBODY DIVERSITY IN THE TURTLE

The Ab response in reptiles has been studied at the protein level, and in turtles some aspects resemble those of cold-blooded vertebrates fr om other classes. The genetic bases for these features are not clear. The present study is the first on the IgH organization and complexity of a reptilian Ig gene system. The approach to cloning turtle (Pseudem ys scripta) sequences is entirely PCR based, and its efficacy is demon strated by obtaining extensive information on a heretofore unexplored Ig gene system. A number of genomic V-H sequences, representing possib ly four families, were isolated, as was a genomic C mu 4 clone. These sequences, used as probes, provided proof that in the turtle there is a single IgH locus with multiple V-H genes and one C mu gene. In North ern hybridizations, the C mu 4 probe detected two transcripts; of the four V-H groups, only one was expressed, and multiple bands indicated the presence of at least two non-p transcripts. Using reverse transcri ption-PCR on spleen or liver RNA, an IgM heavy chain sequence was obta ined, as were a number of VDJ rearrangements. Among 32 unique VDJ rear rangements from one animal, there were 22 sequence variants at framewo rk 4, suggesting either a very large number of J segments or somatic m odification in the variable region. The latter interpretation is suppo rted by point mutations found in framework 3 and CDR3. The number of c hanges is considerably greater than the deduced Tag misincorporation r ate (0.05%).