Organization and Expression of
Immunoglobulin Genes
미생물학교실 권 형 주
B-Cell Development
Generation of antibody diversity
How can all these diversity be generated?- Germ-line theories- Somatic-variation theories
Environment : 107 – 109 개 이상의 antigenic shapes (non-self) Genome 에 존재하는 antibody 이상의 antibody 생산이 요구됨
-Ehrlich side-chain theory : antigen-induced selection
- Instructive hypothesis : flexible antibody molecule acted on by antigen to form a complementary binding site- Dreyer & Bennett : existence of a constant region and a variable region encoded by at least two different genes (??) Tonegawa : somatic recombination somatic mutation
Detection of Ig gene rearrangement by Southern blot hybridization
- Embryonic cell (nonlymphoid cell) 의 Ig DNA- Committed cells of B lymphocyte lineage (adult myeloma cells)
-- VJ rearrangement (pre-B cell) : somatic recombination
Tonegawa’s bombshell-Immunoglobulin gene rearrange
Multigene Organization of Ig Genes
Mouse
Variable-Region Gene Rearrangements
Light-chain DNA undergoes V-J rearrangements
Heavy-chain DNA undergoes V-D-J rearrangements
Mechanism of Variable-Region DNA Rearrangements
- Recombination signal sequences (RSSs) direct recombination
- Gene segments are joined by recombinases
V(D)J recombinase : recombination-activating genes RAG-1 and RAG-2
Circular DNA isolated from thymocytes in which the DNA encoding the chains of the TCR undergoes rearrangement in a process like that involving the Ig genes
- Ig-gene rearrangements may be productive or nonproductive- Allelic exclusion ensures a single antigenic specificity- Allelic exclusion: A single specificity is maintained because only one of the two parental alleles of Ig is expressed by every B cell clone from its earliest maturation stage
Generation of antibody diversity
- Multiple germ-line gene segments- Combinatorial V-J and V-D-J joining generates diversity
- Junctional flexibility adds diversity
- P-addition adds diversity at palindromic sequences- N-addition adds considerable diversity by addition of nucleotides
Somatic hypermutation : Immunoglobulin heavy and light chain genes undergo structural modifications after antigen stimulation
- Antigenic stimulation 후에 발생되는 structural alteration- light chain 과 heavy chain 의 variable region 에서 single base change- Germinal center- cells which have produced a higher-affinity antibody are selected for survival - Dependent on T cells and germinal center - Athymic mice lack T cells and germinal center - no affinity maturation- Affinity maturation
- Somatic hypermutation adds diversity in already-rearranged gene segments
- Immunoglobulin gene diversification differs among species
Class switching among constant-region genes- Switching regions : 2 – 3 kb upstream from each CH segment (except C)
Proposed mechanism for class switching induced by IL-4 in rearranged Ig heavy chain genes
- AID (activation-induced cytidine deaminase) mediates both somatic hypermutation and class switching
- AID : RNA editing enzyme : C U, repair G-C A-T
Experimental demonstration of the role of the enzyme AID in class switching and hypermutation
Expression of Ig Genes- Heavy-chain primary transcripts undergo differential RNA processing : Alternative RNA processing
- Simultaneous expression of IgM and IgD
Synthesis, assembly, and secretion of Igs
Regulation of Ig-Gene transcription- Promoter : AT-rich sequence (TATA box) oct-2 (found only in B cells)- Enhancer : in an orientation-independent manner
- Ig-gene expression is inhibited in T cells
: Ig-gene rearrangement (H, L chain –only in B cells : TCR-gene rearrangement- T cells why????, k-chain 3’ enhancer(3’E) mutation Ig-genePU.1 binding site
Antibody genes and antibody engineering
Human antibody from mice bearing a human artificial chromosome (HAC) that includes entire human heavy- and light-chain loci.
- scFv : single-chain fragment variable- Phage display technology