Organization and Expression
of Immunoglobulin Genes

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)
 Genome에 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 Cd)
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’kE) 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