The earliest bone marrow cell committed to the B-cell lineage is the pro–B cell. Pro–B cells do not produce Ig, but they can be distinguished from other immature cells by the expression of the B lineage–restricted surface molecules CD19 and CD10. RAG1 and RAG2 proteins are first expressed at this stage, and the first recombination of Ig genes occurs in the heavy-chain locus. This recombination brings together one D and one J gene segment, with deletion of the intervening DNA (Fig. 1A). The D segments that are 3′ of the rearranged D segment and the J segments that are 5′ of the rearranged J segment are deleted by this recombination (e.g., D1 and J2–J6 in Fig. 1A). After the D-J recombination event, one of the many 5′ V gene segments is joined to the DJ unit, giving rise to a rearranged VDJ exon. At this stage, all V and D segments between the rearranged V and D gene segments are also deleted. V-to-DJ recombination at the Ig H chain locus occurs only in committed B-lymphocyte precursors and is a critical event in Ig expression because only the rearranged V gene is subsequently transcribed. The TdT enzyme, which catalyzes the addition of junctional N nucleotides (see Fig. 2), is expressed most abundantly during the pro–B stage when VDJ recombination occurs at the IgH locus, and levels of TdT decrease before light-chain gene V-J recombination is complete. Therefore, junctional diversity attributed to addition of N nucleotides is more prominent in rearranged heavy-chain genes than in light-chain genes.
Fig1. Immunoglobulin (Ig) heavy- and light-chain gene recombination and expression. The sequence of DNA recombination and gene expression events is shown for the Ig µ heavy chain (A) and the Ig κ light chain (B). In the example shown in (A), the V region of the µ heavy chain is encoded by the rearranged V1, D2, and J1 gene segments. In the example shown in (B), the V region of the κ chain is encoded by the V2 and J1 gene segments. C, Constant; D, diversity; J, joining; L, leader; mRNA, messenger RNA; V, variable.
Fig2. Junctional diversity. During the joining of different gene segments, addition or removal of nuclo tides may lead to the generation of novel nucleotide and amino acid sequences at the junction. Nucleotides (P sequences) may be added to asymmetrically cleaved hairpins in a templated manner. Other nucleotides (N regions) may be added to the sites of V-D, V-J, or D-J junctions in a nontemplated manner by the action of the enzyme terminal deoxynucleotidyl transferase (TdT). These additions generate new sequences that are not present in the germline. RAG, Recombination-activating gene; RSS, recombination signal sequence; V, variable.
The heavy-chain C region exons remain separated from the newly created VDJ exon by DNA containing the distal J segments and the J-C intron. The rearranged Ig heavy-chain gene is transcribed to produce a primary transcript that includes the rearranged VDJ exon and the Cµ exons. The nuclear RNA of the rearranged heavy-chain gene is cleaved downstream of one of two consensus polyadenylation sites, and multiple adenine nucleotides, called poly-A tails, are added to the 3′ end. This nuclear RNA undergoes splicing, an RNA processing event in which the introns are removed and exons joined together. In the case of the µ RNA, introns between the leader exon and the VDJ exon, between the VDJ exon and the first exon of the Cµ locus, and between each of the subsequent constant region exons of Cµ are removed, thus giving rise to a spliced mRNA encoding the µ heavy chain. If the mRNA is derived from an Ig locus at which rearrangement was productive (in the correct reading frame), translation of the rearranged µ heavy-chain mRNA leads to synthesis of the µ protein. Approximately half of all pro–B cells make productive rearrangements at the Ig H locus on at least one chromosome and can thus go on to synthesize the µ heavy-chain protein. Only cells that make productive rearrangements survive and differentiate further.
Once a productive Ig µ rearrangement is made and the heavy-chain protein is made, the cell differentiates into the pre–B stage. Pre–B cells are developing B-lineage cells that express the Ig µ protein but have yet to rearrange their light chain loci. The pre–B cell expresses the µ heavy chain on the cell surface, in association with other proteins, in a complex called the pre–B-cell receptor, which has several important roles in B-cell maturation.
