414/514 Immunology Principles 415/515 Immunology Principles and Practice

Immunoglobulin Gene Rearrangement

Gene segments are rearranged during B cell maturation to generate the active gene that codes for the antibody combining site a B cell can synthesize

• recognition signal sequences (RSS) present in 5' and 3' ends of each intron separating the Ig gene segments allow the rearrangements to occur at appropriate locations in the genome; there are two RSSs, and their heptamers and nonamers are complementary to one another
  • heptamer...12 (or 23) bp spacer...nonamer
  • nonamer...12 (or 23) bp spacer...heptamer
• RAG1 and RAG2 proteins (encoded in recombination-activation genes) mediate antibody gene rearrangement
  • RAG1 molecule bind RSS nonamers on the 5' and 3' ends of the gene segments to be rearranged, then binds RAG2 forming a RAG1/RAG2 complex that juxtaposes the RSS so they can hybridize with one another, which also generates loops containing the intervening DNA sequence
  • RAG1/RAG2 complex nicks one DNA strand, and the resulting free 3'-OH group attacks the phosphodiester bond (in the complementary DNA strand) that links the RSS to its gene segment, thus forming a "hairpin" loop (this occurs at the junctions between both of the RSS and their contiguous gene segments)
  • DNA repair endonucleases (including RAG1/RAG2 complex, which is a 3'-flap endonuclease (FEN) ... more info on FENs) randomly cleave the hairpin loops, generating unpaired nucleotides on each end, some of which are removed by exonucleases and some of which pair with nucleotides (P additions), generating palindromic blunt ends
  • TdT (terminal deoxynucleotidyl transferase) adds random nucleotides to the blunt ends (N-additions)
  • DNA double-strand break repair (DSBR) enzymes ligate the ends of the gene segments
  • splicing selected DNA sequences to form antibody V-region transcriptional units
    • V-D-J (heavy chain)
    • V-J (light chain)
• antibody genes are activated by this process as a result of two events
  • generation of a novel V region gene by combination of one of many V region gene segments with one of several J gene segments (light chain) or one of several D and one of several J gene segments (heavy chain)
  • strengthening the V region promoter by moving it closer to the enhancer found 3' to the J gene segments on the chromosome
• Production of antibody from activated genes in B cells
  • transcription of rearranged antibody genes generates primary mRNA
  • post-transcriptional processing (splicing by SNRPs) of primary mRNA yields mature mRNA
  • mRNA is capped and a polyA tail is added
  • translation generates the protein
  • transport of translated polypeptides into, and cleavage of the leader region within, the endoplasmic reticulum generates the mature protein
  • assembly, then glycosylation of mature heavy and light chains in endoplasmic reticulum
  • packaging of assembled, glycosylated antibody molecules by the Golgi apparatus
  • transport to cytoplasmic membrane via vesicles
  • integration into cytoplasmic membrane to form BCR (or secretion as antibody if no membrane anchor is present at C-terminus of heavy chain)