The diversity of the adult antibody repertoire is achieved by the random combinatorial association of V, D and J segments, and of the resulting H and L chains, and by significant diversity at the junctions of the gene segments. Junctional diversity is generated both the deletion of variable numbers of nucleotides from the coding regions and by the subsequent addition of non-templated N-region nucleotides at the junctions of Vh, D, and Jh segments. We have recently observed that immunoglobulin (Ig) H chain junctions from newborn spleen and liver displayed a virtual absence of N region nucleotides, whereas 85% of IgH junctions derived from adult spleen cells had N regions. These junctional sequences were derived by preparing RNA or DNA from whole tissues, and amplifying the rearranged V-D- J sequences by PCR. The amplified DNA is cloned and sequenced. We also observed that 80% of the junctions which had no N regions had 1-5 nucleotides which could be encoded by either of the two adjoining gene segments. THis overlap may be a result of pairing of the identical stretches of nucleotides. This concept suggested to us a novel hypothesis to explain the previously observed preferential use of 7183 and Q52 Vh families early in ontogeny. Analysis of all cloned Vh genes leads to the prediction that, in the absence of N regions, Vh7183 and VhQ52 Vh genes will preferentially recombine to D-J segments in frame, while most other Vh families will preferentially recombine out of frame, and therefore be unusable. This bias in the proportion of in frame joins would remain until the appearance of N regions, which would randomize the chance of in frame vs. out of frame joins. It has previously been shown that fetal gammadelta T cells are devoid of N regions, and that TdT levels are low in the thymus until after birth. Therefore, we will also investigate whether the lack of N regions extends to fetal/neonatal cells of the predominant T cell subset, that bearing alphabeta TCR. We will determine when the transition to the adult phenotype (i.e., IgH chains mainly containing N regions) occurs in splenic B cells and in alphabeta thymocytes. To determine if the N- IgH chains are truly generated at a higher rate in neonatal vs. adult B cells, we will analyze junctional sequences from pre-B cells from fetal and newborn tissues and compare them to sequences derived from adult bone marrow pre-B cells. Finally, we will determine if Ly1 B cells, which are thought to be a long lived subpopulation of B cells, maintains the N- phenotype throughout life.
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