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