1. Chapter 5 Organization and Expression of Immunoglobulin Genes Dr. Capers

2. Kuby IMMUNOLOGY Sixth Edition Chapter 5 Organization and Expression of Immunoglobulin Genes Copyright © 2007 by W. H. Freeman and Company Kindt Goldsby Osborne

3.  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?

4.  In germ-line DNA, multiple gene segments code portions of single immunoglobulin heavy or light chain During 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

6.  Dreyer and Bennett – 1965 2 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 region Proposed that there are only single copies of limited classes  Greater complexity was revealed later Light chains and heavy chains (separate multi- gene families) are located on different chromosomes

7.  DNA rearrangement: produces variable region  Later mRNA splicing: produces constant region

9.  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

11. 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

14. 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

15. Mechanism of Variable-Region DNA rearrangements  Catalyzed by enzymes ○ V(D)J recombinase  Proteins mediate V-(D)-J joining ○ RAG-1 and RAG-2

16.  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 http://www.youtube.com/watch?v= AxIMmNByqtM Same transcriptional orientation Opposite transcriptional orientation

19. Allelic Exclusion  Ensures that the rearranged heavy and light chain genes from only 1 chromosome are expressed

21. 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

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

23. 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

24. Ab diversity – P-addition and N-addition

25. 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

26. Class Switching  Isotype switching  After antigenic stimulation of B cell  V H D H J H until combines with C H gene segment  Activation-induced cytidine deaminase (AID) Somatic hypermutation Gene conversion CLASS-SWITCH recombination  IL-4 also involved

27. μ→δ→γ→ε→α IgM→IgD→IgG→IgE→IgA

28. 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

31. 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

32. 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