How is antibody diversity generated?Two early theories:Germline hypothesisThe genome contains many loci encoding antibody molecules. B cells express one of these loci. Different B cells express different loci.Somatic mutation hypothesisThere are a small number of antibody genes which undergo mutation as the B cell matures - thus giving rise to B cells expressing antibody of different specificity.Three genetic loci encode immunoglobulin molecules:- two loci encoding the light chains- kappa locus- lambda locus- one locus encoding the heavy chainThese three loci are located on different chromosomes.The loci encoding immunoglobulins have a unique structure.- composed of “gene segments“The heavy chain locus has multiple V (variable) segments, multiple D (diversity) segments, multiple J (joining) segments and multiple C (constant) segments.During maturation, one of each V, D and J segment is randomly “chosen” and used to encode the final antibody molecule.Germline configuration of the heavy chain locus (mice)Each gene segment may have its own intron-exon structure - e.g. the Cm gene segmentKuby Figure 5-3The kappa locus has a similar structure - BUT - does not have D segments.A kappa chain is encoded by one V segment, one J segment and one C segment.Kuby Figure 5-3The lambda locus also has only V, J and C segments.Kuby Figure 5-3Read Kuby pages 109-110: Multigene Organization of Ig GenesKuby Figure 5-3In heavy chains, the V, D and J segments encode the variable domain while the C segment encodes the constant domain.In light chains, the V and J segments encode the variable domain while the C segment encodes the constant domain.During B cell maturation, immunoglobulin genes undergo rearrangement.During B cell maturation, immunoglobulin genes undergo rearrangement.During B cell maturation, immunoglobulin genes undergo rearrangement.During B cell maturation, immunoglobulin genes undergo rearrangement.The kappa and lambda loci undergo similar rearrangement.Since there are no D segments, there is a single VJ rearrangement.In both heavy and light chain rearrangement, a number of J segments may remain between the V(D) and C segments.These are included in the RNA transcript but are spliced out during RNA processing.The final light chain mRNA contains one VJC unit.Kuby Figure 5-4Kuby Figure 5-4In the case of the heavy chainIn naive, mature B cells, the primary mRNA transcript contains VDJ and BOTH Cm and Cd.This primary transcript can be differentially processed to give rise to mRNA encoding either IgM or IgD.This explains why naive B cells express both IgM and IgD.The variable region of both are encoded by the same VDJ - so they have the same antigen specificity.Kuby Figure 5-5Read Kuby pages 110-112: Variable-Region Gene RearrangementsWhat mechanism ensures correct joining of gene segments during rearrangement of the heavy and light chain loci?Kuby Figure 5-3Recombination signal sequences - conserved sequences in regions just upstream or downstream of gene segments.Consist of a conserved heptamer and nonamer with a 12 or 23 bp spacer.Kuby Figure 5-3What mechanism ensures correct joining of gene segments during rearrangement of the heavy and light chain loci?Recombination signal sequences - conserved sequences in regions just upstream or downstream of gene segments.Consist of a conserved heptamer and nonamer with a 12 or 23 bp spacer.The one-turn/two-turn rule (12/23 rule) - recombination occurs only between a segment with a 12 bp spacer and a segment with a 23 bp spacer.The one-turn/two-turn rule (12/23 rule) - recombination occurs only between a segment with a 12 bp spacer and a segment with a 23 bp spacer.Read Kuby page 113: Recombination Signal Sequences Direct RecombinationKuby Figure 5-6Recombination Activating Genes (encode RAG-1 and RAG-2)RAG-1 and RAG-2 mediate recognition of signal sequences and rearrangement of DNA segments.Mice deficient in RAG-1 or RAG-2 are unable to rearrange heavy or light chain genes.These mice have no mature B cells. B cells will not mature if they cannot express a BCR.RAG-deficient mice also lack mature T cells. The T cell receptor is also encoded by loci containing gene segments that must be rearranged before the TCR can be expressed.T cells will not mature if they cannot express a TCR.SCID mice also have a defect that affects rearrangement of BCR and TCR loci. They also have no mature T or B cells.Flow cytometry of normal vs. RAG-1 deficient mice:Lymph node cellsFITC anti-CD19 (B cell marker) and PE anti-CD3 ( T cell marker)Normal miceRAG-1 - deficient mice533553A G C TT A T ATerminal deoxynucleotidyl transferase (Tdt)An enzyme that randomly adds in nucleotides during joining of heavy chain (NOT light chain) segments.Read Kuby pages 113-115: Gene Segments Are Joined by RecombinasesProductive and nonproductive rearrangementsJoining of segments is not precise and may result in loss of the correct reading frame.This may lead to introduction of stop codons nonpr