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,
,
*
The Hooper Foundation, San Francisco, CA 94143; and
Ontario Cancer Institute, and
Department of Immunology, University of Toronto, Toronto, Ontario, Canada
Ig diversity is generated in large part by the combinatorial joining of the Ig gene segments, VH, D, and JH, that together encode the variable domain of Ig. The final Ig repertoire, however, not only reflects the diversity generated through V(D)J recombinatorial joining, but it is also the product of a number of developmental restraints and selections. To avoid such restrictions and assess the recombination potential of individual Ig gene segments, we constructed Ig heavy (H) chain microlocus plasmids, each of which contain germline coding, recombination signal, and flanking sequences of a VH, D, and JH gene segment. These plasmids allow us to assess the recombination potential of the segments in the context of their natural flanking DNA sequences, but in the absence of any higher order chromatin structure or cellular selection. We found that the frequency and extent of deletions and additions at the recombination breakpoints are similar to those observed at rearranged Ig H chain loci in intact animals. The relative frequencies of the types of rearrangements—VD-J, V-DJ, VinvD-J (invD = inverted D), and VDJ—however, differ strongly. Moreover, V81x, the most used VH gene segment in intact mice, also is overused in this plasmid assay, 15 to 30 times that of another VH segment. This result indicates that the overuse of V81x in the early B cell repertoire can be a consequence of its DNA sequence and not of cellular activities.
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