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Multiple Sites of V Diversification in Cattle¹

Mark R. Lucier, Rachel E. Thompson^*, James Waire, Athena W. Lin, Barbara A. Osborne^, and Richard A. Goldsby^2,,*,

^* Department of Biology, Amherst College, Amherst, MA 01002; and Department of Veterinary Sciences and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003.

Ig repertoire diversification in cattle was studied in the ileal Peyer’s patch (IPP) follicles of young calves and in the spleens of late first-trimester bovine fetuses. To investigate follicular diversification, individual IPP follicles were isolated by microdissection; V diversity was examined by RT-PCR and subsequent cloning and sequencing. When 52 intrafollicular sequences from a 4-wk-old calf were determined and compared, two major groups, one of 23 members and the other of 25, could be delineated. An examination of these groups revealed clear genealogic relationships that implicated in situ diversification of V sequences within the confines of an IPP follicle. V expression was also examined in early (95 and 110 gestational day) fetal bovine spleens. Although earlier studies in cattle and sheep implicated the IPP as a likely site of Ab diversification, a close investigation of V sequences in late first-trimester fetal calves revealed that diversity appears in the early fetal spleen before the establishment of a diverse repertoire in the ileum. When the sequences for the fetal spleen were compared with an existing pool of germline sequences, we found evidence of possible gene conversion events and possible untemplated point mutations occurring in sequences recovered from fetal spleens. We conclude that IPP is not the sole site of V diversification in cattle. Also, as suggested for rabbits, cattle may use both gene conversion and untemplated somatic point mutation to diversify their primary V repertoire.

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