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FIGURE 8-8 Antigen receptor gene rearrangements.

Southern blot analysis of DNA from nonlymphoid (liver) cells and from a monoclonal population of B lymphocyte lineage origin (e.g., a B cell tumor) is shown in schematic fashion. The DNA is digested with a restriction enzyme (EcoRI as depicted), different-sized fragments are separated by electrophoresis, and the fragments are transferred onto a filter. The sites at which the EcoRI restriction enzyme cleaves the DNA are indicated by arrows. The size of the fragments containing the JK3 segment of the Ig k light chain gene or the VK29 V region gene was determined by use of a radioactive probe that specifically binds to JK3 segment DNA or to VK29 DNA. In the hypothetical example shown, VK29 is part of a 5-kb EcoRI fragment in liver cells but is on a 3-kb fragment in the B cell clone studied. Similarly, the JK3 fragment is 8 kb in liver cells but 3 kb in the B cell clone.

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FIGURE 8-8 Antigen receptor gene rearrangements.

Southern blot analysis of DNA from nonlymphoid (liver) cells and from a monoclonal population of B lymphocyte lineage origin (e.g., a B cell tumor) is shown in schematic fashion. The DNA is digested with a restriction enzyme (EcoRI as depicted), different-sized fragments are separated by electrophoresis, and the fragments are transferred onto a filter. The sites at which the EcoRI restriction enzyme cleaves the DNA are indicated by arrows. The size of the fragments containing the JK3 segment of the Ig k light chain gene or the VK29 V region gene was determined by use of a radioactive probe that specifically binds to JK3 segment DNA or to VK29 DNA. In the hypothetical example shown, VK29 is part of a 5-kb EcoRI fragment in liver cells but is on a 3-kb fragment in the B cell clone studied. Similarly, the JK3 fragment is 8 kb in liver cells but 3 kb in the B cell clone.

a single rearrangement event joins a randomly selected

V gene to an equally randomly selected J segment. The IgH and TCR P and 8 loci contain D segments, and at these loci two distinct rearrangement events must be separately initiated, first joining a D to a J and then a V segment to the fused DJ segment. Each rearrangement event involves a number of sequential steps. First, the chromatin must be opened in specific regions of the antigen receptor chromosome to make gene segments accessible to the enzymes that mediate recombination. Next, two selected gene segments must be brought next to one another across a considerable chromosomal distance. Double-stranded breaks are then introduced at the coding ends of these two segments, nucleotides are added or removed at the broken ends, and finally the processed ends are ligated to produce clonally unique but diverse antigen receptor genes that can be efficiently transcribed. The C regions lie downstream of the rearranged V(D)J exon separated by the germline J-C intron. This rearranged exon is transcribed to form a primary (nuclear) RNA transcript. Subsequent RNA splicing brings together the leader exon, the V(D)J exon, and the C region exons, forming an mRNA that can be translated on membrane-bound ribosomes to produce one of the chains of the antigen receptor. The use of different combinations of V, D, and J gene segments and the addition and removal of nucleotides at the joints contribute to the tremendous diversity of antigen receptors, as we will discuss in more detail later.

Recognition Signals That Drive V(D)J Recombination

Critical lymphocyte-specific factors that mediate V(D)J recombination recognize certain DNA sequences called recombination signal sequences (RSSs), located 3' of each

V gene segment, 5' of each J segment, and flanking each D segment on both sides (Fig. 8-10A). The RSSs consist of a highly conserved stretch of 7 nucleotides, called the heptamer, usually CACAGTG, located adjacent to the coding sequence, followed by a spacer of exactly 12 or 23 nonconserved nucleotides, followed by a highly

Germline DNA

V1 V2 Vn

D1-n

J1-n

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