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justin.ppt justin.ppt Presentation Transcript

  • The Organization and Expression of Ig Genes Justin Walter Immunology, Fall 2008
  • Introduction  The vertebrate immune system is capable of responding to an essentially infinite array of foreign antigens  Variable vs. constant regions  Basis for variability  organization and expression of Ig genes
  • Overview  Historical perspective – early theories  “Multigene” organization of Ig genes  Variable region gene rearrangements  Basics of mechanism – key players: DNA signal sequences, specific enzymes  Generation of Ab diversity  seven primary routes
  • Historical perspective  Ig sequence analysis revealed many dilemmas  Extreme diversity of Ab specificity  Variable regions vs constant regions  Isotypes with similar Ag specificity but differing heavy-chain constant regions
  • Historical perspective  Proponents of the one-gene-one-protein paradigm had trouble reconciling this model with the oddities of Igs.  This led to an initial “germ-line theory” which suggested that a significant portion of the genome is dedicated solely to Ab coding.  Argument: the immune system is THAT important
  • Historical perspective  In contrast, “somatic-variation theories” emerged which suggested the opposite:  Relatively small amount of Ig genes  Specificity arises from mutation and/or recombination
  • Historical perspective  Dreyer & Bennet (1965)  Two-gene, one-protein model  No precedent in any biological system
  • Historical perspective  Tonegawa & Hozumi (1976)  Compare Ig DNA from embryonic (germline) and adult myeloma (somatic) cells  Experimental data suggested that during differentiation, the V and C genes undergo rearrangement.  1987 Nobel prize
  • Tonegawa & Hozumi
  • Multigene Organization of Ig genes  Each “class” of Ig components (kappa, lambda, heavy) encoded by separate multigene families on different chromosomes  Each family contains several coding sequences, or gene segments
  • Multigene Organization of Ig genes
  • Multigene Organization of Ig genes  Κ & λ light chains: V (variable), J (joining), and C (constant) gene segments  Heavy chains: V, D (diversity), J, and C gene segments  A leader (L) sequence also precedes each V segment.  Gene segments discovered by comparing DNA sequences with amino acid sequences of Igs  Tonegawa, again.
  • Organization of Ig germ-line gene segments (mouse) Pre-rearrangement!
  • Variable-region rearrangement  Multifaceted process, produces mature B cells which are “committed” to express specific Ab  Specificity of Ab determined by the sequence of its rearranged variable genes.
  • Light-chain rearrangements  V-J rearrangements  Specific “allowed” rearrangements differ from species to species, but a “big-picture” view can suffice  Rearrangement occurs in ordered steps but can be considered as random events which result in the random determination of Ab specificity
  • Kappa light-chain rearrangement & RNA processing Leader sequence targets nascent protein to ER and is subsequently cleaved
  • Heavy-chain rearrangements  Requires two separate rearrangement events  D-Jjoining  V-DJ joining  Differential polyadenylation & RNA splicing can result in mRNA with either Cu or Cδ  heavy chain genes B cells can express BOTH IgM and IgD with identical Ag specificity on its surface
  • Heavy-chain rearrangement The focus here is on IgM IgD B cells…
  • Mechanism of Variable region DNA rearrangements  Recombination signal sequences direct recombination
  • Mechanism of Variable region DNA rearrangements  Recombination signal sequences direct recombination Vλ, JK, VH, JH VK, Jλ, DH
  • Mechanism of Variable region DNA rearrangements  A one-turn RSS can only join with a two- turn RSS  Why might this be?
  • Mechanism of Variable region DNA rearrangements  Gene segments are joined by a class of enzymes called recombinases  Two recombination-activating genes encode proteins which act together to mediate V-(D)-J joining  RAG-1  RAG-2
  • a. Same b. opposite transcriptional transcriptional orientation orientation (most common) Addn. of P- nucleotides accomplished with repair enzymes
  • ?
  • Mechanism of Variable region DNA rearrangements  Rearrangements may be productive or nonproductive.
  • Mechanism of Variable region DNA rearrangements  Allelic exlusion  Heavy-chain genes only expressed from one chromosome  Light-chain genes only expressed from one chromosome  Essential for specificity  Expression of both alleles would result in a “multispecific” B cell
  • Mechanism of Variable region DNA rearrangements  Allelic exlusion
  • Generation of Ab diversity  Multiple germ-line gene segments  Combinatorial V-(D)-J joining  Junctional flexibility  P-region nucleotide addition  N-region nucleotide addition  Somatic hypermutation  Combinatorial association of light and heavy chains
  • Possibly as high as 1010!
  • Junctional flexibility
  • P/N-addition
  • Somatic hypermutation  Nucleotide replacement, mediated by activation-induced cytidine deaminase (AID)  Also plays a key role in class switching  Frequency of 10-3 per bp per generation  100,000X the rate of spontaneous mutation!  Approx 1 mutation every 2 cell divisions
  • Somatic hypermutation