Chapter 7 Organization and Expression of Immunoglobulin Genes Dr. Capers.

Chapter 7Organization and Expression of Immunoglobulin

GenesDr. Capers

Kuby IMMUNOLOGYSixth Edition

Chapter 5Organization and Expression of

Immunoglobulin Genes

Copyright © 2007 by W. H. Freeman and Company

Kindt • Goldsby • Osborne

How does antibody diversity arise?

What causes the difference in amino acid sequences?

How can different heavy chain constant regions be associated with the same variable regions?

In germ-line DNA, multiple gene segments code portions of single immunoglobulin heavy or light chainDuring B cell maturation and

stimulation, gene segments are shuffled leaving coding sequence for only 1 functional heavy chain and light chain○ Chromosomal DNA in mature B cells is not

the same as germ-line DNA

Dreyer and Bennett – 19652 separate genes encode single

immunoglobulin heavy or light chain○ 1 for the variable region

Proposed there are hundreds or thousands of these

○ 1 for the constant regionProposed that there are only single copies of limited

classes

Greater complexity was revealed laterLight chains and heavy chains (separate

multi-gene families) are located on different chromosomes

DNA rearrangement: produces variable region

Later mRNA splicing: produces constant region

Kappa (κ) and lamda (λ) light chain segments:○ L – leader peptide, guides through ER○ V VJ segment codes for variable region

○ J○ C – constant region

Heavy chain○ L ○ V VDJ segment codes for variable region

○ D○ J○ C

Variable-region gene rearrangements

Variable-region gene rearrangements occur during B-cell maturation in bone marrow○ Heavy-chain variable region genes rearrange

first○ Then light-chain variable region○ In the end, B cell contains single functional

variable-region DNA sequence

○ Heavy chain rearrangement (“class switching”) happens after stimulation of B cell

Mechanism of Variable-Region DNA rearrangements Recombination signal sequences

(RSSs)○ Between V, D, and J segments○ Signal for recombination○ 2 kinds

- 12 base pairs (bp) – 1 turn of DNA- 23 bp – 2 turns of DNA- 12 can only join to 23 and vice versa

Mechanism of Variable-Region DNA rearrangements

Catalyzed by enzymes○ V(D)J recombinase

Proteins mediate V-(D)-J joining○ RAG-1 and RAG-2

Gene arrangements may be nonproductive○ Imprecise joining can occur so that reading frame is not complete○ Estimated that less than 1/9 of early pre-B cells progress to

maturity

Gene rearrangement video: http://www.youtube.com/watch?v=AxIMmNByqtM

Look at Figure 7-8 – VDJ recombination○ 1. Recognition of RSS by RAG1/RAG2 enzyme complex○ 2. One-strand cleavage at junction of coding and signal sequences○ 3. Formation of V and J hairpins and blunt signal end○ 4. ligation of blunt signal end to form signal joint

- 2 triangles on each end (RSS) are joined○ 5. Hairpin cleavage of V and J regions○ 6. P nucleotide addition (palindromic nucleotide addition – same if

read 5’ to 3’ on one strand or the other○ 7. Ligation of light V and J regions (joining)○ 8. Exonuclease trimming (in heavy chain)

- Trims edges of V region DNA joints

○ 9. N nucleotide addition (non-templated nucloetides)○ 10. Ligation and repair

Allelic Exclusion

Ensures that the rearranged heavy and light chain genes from only 1 chromosome are expressed

Generation of Antibody 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

○ This is mainly in mice and humans – other studied species differ in development of diversification

Ab diversity – Multiple gene-line segments AND combination of those segments

Ab diveristy – junctional flexibility Random joining of V-(D)-J segments

○ Imprecise joining can result in nonproductive rearrangements

○ However, imprecise joining can result in new functional rearrangements

Ab diversity – P-addition and N-addition

Ab diversity – somatic hypermutation Mutation occurs with much higher

frequency in these genes than in other genes

Normally happens in germinal centers in lymphoid tissue

Class Switching Isotype switching After antigenic stimulation of B cell VHDHJH until combines with CH gene

segment Activation-induced cytidine deaminase

(AID)Somatic hypermutationGene conversionCLASS-SWITCH recombination

IL-4 also involved

μ→δ→γ→ε→αIgM→IgD→IgG→IgE→IgA

Ig Gene Transcripts

Processing of immunoglobulin heavy chain primary transcript can yield several different mRNAs○ Explains how single B cell can have

secreted and membrane bound Ab

Regulation of Ig-Gene Transcription 2 major classes of cis regulatory sequences in DNA

regulate Promoters – promote RNA transcription in specific

direction Enhancers – help activate transcription

Gene rearrangement brings the promoter and enhancer closer together, accelerating transcription

Antibody Engineering Monoclonal Abs used for

many clinical reasons (anti- tumor Ab, for instance)

If developed in mice, might produce immune response when injected

○ Can be cleared in which they will not be efficient

○ Can create allergic response

Creating chimeric Abs or humanized Abs are beneficial

Rearrangement of TCR genes Similar to that of Ig

Rearrangement of α and γ chains○ V, J, and C segments

Rearrangement of β and δ chains○ V, D, J, and C segments

Generation of TCR diversity (a lot like Ig)○ Multiple germ-line gene segments○ Combinatorial V-(D)-J joining○ Junctional flexibility○ P-region nucleotide addition○ N-region nucleotide addition○ Combinatorial association of light and heavy

chains However, there is no somatic

mutation with TCR○ May be to ensure that after thymic

selection, the TCR doesn’t change to cause self-reactive T cell