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Chapter 5
Organization and Expression of
Immunoglobulin Genes
Genetic Models
- How to account for :
- Vast diversity of antibody specificities
- Presence of Variable regions at the amino
end of Heavy and Light chains, and a
Constant region at the carboxyl end
- Existence of isotypes (different Heavy
chains) with same antigenic specificity
Models to Explain Antibody Diversity
- The Germ Line Theory : “genome posses the large repertoire of antibody genes to
account for all the antibody diversity”
- The Somatic Variation Theory : “genome
posses a relatively small number of antibody genes and diversity is generated by
mutation and recombination of these genes during somatic development”
The two-gene model:
- Developed by Dreyer and Bennet in 1965
- Two separate genes code for the Heavy
and Light chains. One codes for the V
region and the other for the C region
- These genes come together during at the
DNA level to form a continuous message
- There are thousands of V genes in germ
line but only one gene for the C region
Tonegawa (1976): Immunoglobulin gene rearrangement
- J Probe
- Digested fragments
Three genetic loci encode immunoglobulin molecules:
- Two loci encoding the light chains
- One locus encoding the heavy chain
These three loci are located on different chromosomes.
Multigene Families
- Light Chains : V, J and C gene segments.
- Lambda : Humans (30V, 4J and 7C genes)
- Kappa : Humans (40V, 5J and 1C genes)
- Heavy Chains : V, D , J and C gene segments
- Heavy Chains : Humans (50V, 25D, 6J and 8 C genes)
- In heavy chains, the V, D and J segments encode the variable domain while the C segment encodes the constant domain.
- In light chains, the V and J segments encode the variable domain while the C segment encodes the constant domain.
The loci encoding immunoglobulins have a unique structure.
- composed of "gene segments"
- The heavy chain locus has multiple V (variable) segments, multiple D (diversity) segments, multiple J (joining) segments and multiple C (constant) segments.
During maturation, one of each V, D and J segment is randomly “chosen” and used to encode the final antibody molecule.
Germline configuration of the heavy chain locus (mice)
1, rearrangement – D7/J
6, Post-translational modifications
5, polypeptide
4, mature mRNA
1, rearrangement – D7/J3 and V
3, Post-transcriptional modifications
2, RNA transcript
Kuby Figure 5-
Gene rearrangement Kappa light Chain
The hairpin is cut at a random site…
Signal Joint
Endonuclease (Artemis)
Coding Joint
A G C T
T A T A
Palindromic sequences may form…
Terminal deoxynucleotidyl transferase (Tdt)
An enzyme that randomly adds in nucleotides during joining of coding gene segments.
P-nucleotides
The join is repaired…
Coding Joint
Note: Keep in mind that this random rearrangement can lead to PRODUCTIVE and NON-PRODUCTIVE gene rearrangements
V P N P D P N P J C
V P N P J C
Heavy chain:
Light chain:
The final “gene” encoding the antibody produced by a B cell (and T cells) consists of a number of different segments.
This process of recombination of different gene segments and addition of P and N nucleotides ensures that an enormous number of different antigen specificities are possible.
Generation of antibody diversity
1. Multiple germline V, D and J gene segments
2. Combinatorial V-J and V-D-J joining
3. Somatic hypermutation
4. Junctional flexibility
5. P-nucleotide addition
6. N-nucleotide addition
7. Combinatorial association of heavy and light chains
Combinatorial V-J and V-D-J joining
- Humans:
- Heavy Chain: V (51), D (27), J (6) = 8262
- Light Chain: Kappa – V (40), J (5) = 200
Lambda – V(30), J (4) = 120
8262 x (200 x 120) = 2.64 x 10^6
Junctional flexibility
- Generated through V, D and J combinations
- Joining of Recombination Signal Sequences = Signal Joint
- Joining of Coding Sequences = Coding Joint
- Signal Joints ALWAYS joined precisely, but joining of Coding Joints is IMPRECISE
- Good = Antibody diversity
- BAD = Non=productive rearrangements
Imprecise Always Precise
Additions/Deletions
P-nucleotide addition
- Cleavage of the Hairpin at the end of the coding sequence by endonuclease (Artemis) is random
- Generates a short single strand of nucleotides at the end of the Coding sequence
- Addition of complementary nucleotides to this strand forms a palindrome sequence (P nucleotides)
Generation of antibody diversity
1. Multiple germline V, D and J gene segments
2. Combinatorial V-J and V-D-J joining
3. Somatic hypermutation
4. Junctional flexibility
5. P-nucleotide addition
6. N-nucleotide addition
7. Combinatorial association of heavy and light chains
2.64 x 10^6 to 7.2 x x 10^7 variabilities!!!!
ALLELIC EXCLUSION:
- We have two copies (alleles) of each Ig gene - one
inherited from our father and one from our mother.
- In most cases, both genes are expressed.
- But Antibody genes are different! …. Only one heavy
chain allele and one light chain allele is expressed!!!
- This is termed allelic exclusion (one allele is
excluded). Once a productive arrangement is made,
the other allele is suppressed
- Why? To ensure that each B cell makes antibody of
a single specificity.
- Ig genes are located in 3 chromosomes
- Allelic exclusion
κκκκ λλλλ HH
Pro-B cell Pre-B cell Immature B cell
Class Switching
- Antigen stimulation of a B cells Antibodies with same variable Heavy (VDJ) with any CH gene segment
- Process dependent on Switch Regions
- Switch Regions (2-3 kb) are located upstream from each CH segment, except IgD (Cδ)
- Process driven by cytokines:
- IL-4 IgM to IgG1 or IgE
- IFN-γ IgM to IgG2a
- Players in regulation: 1) switch regions, 2) switch recombinases, 3) cytokine signals
Class Switching
IL-
IgM IgG
- Recombination between Switch Regions
- Switching only proceeds downstream
IgM IgG
Note: Same specificity but different H chain
SAME!!!
AID Enzyme
- Activation induced cytidine deaminase
- RNA editing enzyme
- Deamination of cytosine uracyl repair
induces base modifications!!!
- Mediates SOMATIC HYPERMUTATION,
GENE CONVERSION, and CLASS switching recombination
- No class switching
- Little or no point mutations
Expression of membrane or secreted
Immunoglobulin
- In mature B cells membrane forms; in Plasma
cells secreted forms
- Process depends on differential processing of
primary transcript
- Remember: IgG, IgD, IgA (3 CH domains), IgM and IgA (4 CH domains).
- Domain 3/4 contains the Secretory (hydrophilic)
nucleotide sequence (S) at its 3’.
- Two Exons at 3’ encode the M1 (trans-membrane) and
M2 (cytoplasmic) segments.
- Primary transcript contains two PolyA sites: If
cleavage at Poly A site I = Secreted Form. If cleavage
at PolyA site 2 = Membrane Form
The End
strand of DNA
- This occurs at the border of
the RSS heptamer and the
coding gene
- The 3’ OH group attacks a
phosphodiester bond on
the other DNA strand
- This results in hairpin DNA
strand on the coding
region.
- Other enzymes get involved
and remove the “junk” and
bring together the coding
regions
V DJ
- Terminal deoxynucleotidyl transferase (TdT) is important for creating junctional diversity
- What are SIGNAL JOINTS and CODING JOINTS?
- Hairpin must be opened the enzyme Artemis
- Cleavage is random and can happened at any site in the hairpin
- Replication results in a short inverted repeat or palindrome = P nucleotides
- TdT now can introduce random nucleotides into the coding joints = N nucleotides
- Keep in mind that all this introduced variability may results in functional and non-functional Ig (or TCR) genes.
Junctional Diversity:
IgM IgG
IgM IgE
