The Organization and

Expression of Ig
Genes

Introduction
The vertebrate immune system is capable
of responding to an essentially infinite
array of foreign antigens
Variable vs. constant regions
Basis for variability organization and
expression of Ig genes

Overview
Historical perspective early theories
Multigene organization of Ig genes
Variable region gene rearrangements

Basics

of mechanism key players: DNA

signal sequences, specific enzymes

Generation of Ab diversity
seven

primary routes

The Conundrum

The human genome contains an estimated

20,000 to 25,000 genes that encode mRNAs
for proteins.
To protect us, our immune system has the
ability to produce about 1010-1011 different
antibodies.
If every gene encoded a different antibody,
the genes would encode only 1 millionth of
the antibodies needed.
How can our immune system produce so
many different antibodies with so few genes?

Objectives

Understand how the immunoglobulin genes are

organized in the DNA.
Understand the basis for diversity of
immunoglobulins.
Understand the mechanisms by which the
variable regions undergo gene rearrangement.
Understand the mechanism of class switching.
Understand how immunoglobulin genes are
expressed.
Understand the medical consequences of the
activity of genetic rearrangement mechanisms.

The Keys to Antibody Diversity

Antibody diversity is generated during genetic

rearrangement by mixing and matching one of each
of the various gene segments for the heavy and light
chains in a combinatorial manner.
Antibody diversity is generated by errors incorporated
at the joining sites for the various segments of the
heavy and light chains.
Antibody diversity is generated by hypermutation in
one of the gene segments (variable regions) of the
heavy and light chains during proliferation of B cells.

Historical perspective

Ig sequence analysis revealed many

dilemmas
Extreme

diversity of Ab specificity
Variable regions vs constant regions
Isotypes with similar Ag specificity but differing
heavy-chain constant regions

Historical perspective

Proponents of the one-gene-one-protein

paradigm had trouble reconciling this model with
the oddities of Igs.
This led to an initial germ-line theory which
suggested that a significant portion of the
genome is dedicated solely to Ab coding.
Argument:

the immune system is THAT important

Historical perspective

In contrast, somatic-variation theories

emerged which suggested the opposite:
Relatively

small amount of Ig genes

Specificity arises from mutation and/or
recombination

Historical perspective

Dreyer & Bennet (1965)

Two-gene,

one-protein model
No precedent in any biological system

Historical perspective

Tonegawa & Hozumi (1976)

Compare

Ig DNA from embryonic (germline)

and adult myeloma (somatic) cells
Experimental data suggested that during
differentiation, the V and C genes undergo
rearrangement.
1987 Nobel prize

Tonegawa & Hozumi

Multigene Organization of Ig genes

Each class of Ig components (kappa,

lambda, heavy) encoded by separate
multigene families on different
chromosomes
Each

family contains several coding

sequences, or gene segments

Multigene Organization of Ig genes

Multigene Organization of Ig genes

& light chains: V (variable), J (joining), and C

(constant) gene segments
Heavy chains: V, D (diversity), J, and C gene
segments
A leader (L) sequence also precedes each V
segment.
Gene segments discovered by comparing DNA
sequences with amino acid sequences of Igs
Tonegawa,

again.

Organization of Ig germ-line
gene segments (mouse)

Pre-rearrangement!

Variable-region rearrangement

Multifaceted process, produces mature B

cells which are committed to express
specific Ab
Specificity

of Ab determined by the sequence

of its rearranged variable genes.

Light-chain rearrangements

V-J rearrangements
Specific

allowed rearrangements differ from

species to species, but a big-picture view
can suffice
Rearrangement occurs in ordered steps but
can be considered as random events which
result in the random determination of Ab
specificity

Kappa light-chain
rearrangement & RNA
processing
Mouse: 85V:5J:1C
Human: 40V:5J:1C

Leader sequence targets

nascent protein to ER
and is subsequently
cleaved

Heavy-chain rearrangements

Requires two separate rearrangement events

D-J

joining
V-DJ joining

Differential polyadenylation & RNA splicing can

result in mRNA with either Cu or C
heavy chain genes
B

cells can express BOTH IgM and IgD with identical

Ag specificity on its surface

Heavy-chain
rearrangement
51V:27D:6J

IgM

IgD

The focus here is on

B cells

How Does Rearrangement

Occur?
Rearrangement occurs between specific
sites on the DNA called recombination
signal sequences (RSSs).
Rearrangement is catalyzed by two
recombination-activating genes: RAG-1
and RAG-2.

Mechanism of Variable region

DNA rearrangements
Gene segments are joined by a class of
enzymes called recombinases
Two recombination-activating genes
encode proteins which act together to
mediate V-(D)-J joining

RAG-1
RAG-2

Mechanism of Variable region

DNA rearrangements

Recombination signal sequences direct recombination

Mechanism of Variable region

DNA rearrangements

Recombination signal sequences direct recombination

V, JK, VH, JH

VK, J, DH

Rearrangement Process
Site of P and N nucleotide
additions

Recombination Signal Sequences

The specific recognition sequences, called recombination signal

sequences (RSSs), indicate the sites of recombination.
These signal sequences composed of a 7 bp sequence and a 9 bp
sequence are separated by one turn of the DNA (12 bp spacer) or by
two turns of the DNA (23 bp spacer).
The RSSs are inverted repeats that allow the DNA to form a stemloop with the RSSs aligning on the stem.

Mechanism of Variable region

DNA rearrangements

A one-turn RSS can only join with a twoturn RSS

Why

might this be?

2..1
1..2
2.1..1.2

a. Same
transcriptional
orientation
(most common)

Addn. of Pnucleotides
accomplished
with repair
enzymes

b. opposite
transcriptional
orientation

Mechanism of Variable region

DNA rearrangements

Rearrangements may be productive or

nonproductive.

When gene segments join

in phase, reading frame is
maintained.- productive
When gene segments join
out of phase, reading frame is
is not maintained-nonproductive

Consequences of Rearrangement and

P- and N-Nucleotide Addition

Positive consequence of imprecise joining of Ig gene

segments

A productive rearrangement occurs if the number of nucleotides

added across the joining region allows the genetic code to be read
in phase.
This results in the generation of additional diversity.

Negative consequence of imprecise joining of Ig gene

segments

A nonproductive rearrangement occurs if the number of

nucleotides added across the joining region causes the genetic
code to be read out of phase.
This results in an incomplete antibody (run into stop codons)
The B cell may be able to productively rearrange the
immunoglobulin gene on the other chromosome.
Otherwise, it will result in the death of the B cell.

Mechanism of Variable region

DNA rearrangements

Allelic exlusion
Heavy-chain

genes only expressed from one

chromosome
Light-chain genes only expressed from one
chromosome

Essential for specificity

Expression

of both alleles would result in a

multispecific B cell

Alleleic Exclusion
Alleleic exclusion occurs when only one of two
alleles is expressed.
This is the case with immunoglobulin
molecules.
This ensures that a given B cell will make
antibody molecules with only a single
specificity.
Antibodies may be made from maternal and
paternal chromosomes:HM:LM; HP:LM; HM:LP;
HP:LP.
Note: If the first allele makes a non-functional
antibody, the second allele will undergo
rearrangement.

Mechanism of Variable region

DNA rearrangements

Allelic exlusion

Generation of Ab 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

Possibly as high as 1010!

Junctional flexibility

P/N-addition

Somatic hypermutation

Nucleotide replacement, mediated by

activation-induced cytidine deaminase
(AID)
Also

plays a key role in class switching

Frequency of 10-3 per bp per generation

100,000X

the rate of spontaneous mutation!

Approx 1 mutation every 2 cell divisions

Somatic hypermutation

Immunoglobulin Molecules
Expressed on Mature B Cells

Mature (but not activated) B cells initially express IgD

and IgM on their external cell membranes.

As mature B cells are activated to divide and differentiate

by their cognate antigen, they switch from membranebound IgD and IgM to secretory IgM.

The choice of IgD versus IgM occurs at the level of processing of

mRNA, so a given B cell can both express IgD and IgM.

This switch occurs at the level of processing of mRNA

transcripts.

As they continue to divide and differentiate, they may

undergo additional class switching: IgM => IgG => IgE
=> IgA.

These switches occur at the level of rearrangements of the DNA.

Expression of Membrane-Bound IgD and IgM

An initial pre-mRNA
transcript is produced.
Importantly, the premRNA transcript has
two poly-A sites.
If the second
polyadenylation site is
read, then the mRNA
for membrane-bound
IgM is generated by
splicing.
If the fourth
polyadenylation site is
read, then the mRNA
for membrane-bound
IgD is generated by
alternate splicing.

Structures of Membrane IgM and Secreted IgM

Note the cysteine

for creation of S-S
bonds between IgM
antibodies.

Note the many

hydrophobic
amino acids in
the transmembrane
sequence.

Expression of MembraneBound IgM and Secreted IgM

The initial premRNA transcript is

synthesized.
Importantly, the
pre-mRNA
transcripts have
two poly-A sites
within the C gene
segment.
If the M1, M2
exons are spliced
out, the mRNA for
secreted IgM is
produced.

Class Switching With Activated B

Cell Differentiation And Division

After activation, B cells switch from

membrane-bound IgM and IgD to secreted
IgM by differential splicing.
As the activated B cells continue to
differentiate and divide, they class switch to
production of IgG by DNA rearrangement.
Activated B cells may continue to class switch
to production of IgE or IgA by DNA
rearrangement.

Mechanism of Class
Switching
With activation of
the B cell, class
switching can occur.
At the level of the
DNA, a looping
event occurs that
cuts out the
constant regions for
IgM and IgD.
This leads to the
production of IgG
mRNAs.
If further looping out
occurs, the mRNAs
for IgE or IgA are
produced.

Cytokine Effects on Class

Switching
Certain cytokines
affect class
switching:
IFN- => IgG2a
IL-4 => IgG1, IgE
IL-5 => IgE

Antibody Diversity by Hypermutation

After exposure to its cognate

antigen, the mature B cell is
activated to proliferate.
As the B cell proliferates,
mutations accumulate in the
immunoglobulin gene by a
process called somatic
hypermutation.
These mutations are
concentrated in the variable
region.
The mutations give greater
antibody diversity.
Some of the mutations will
lead to an antibody that
binds the antigen more firmly
(affinity maturation).
If the mutations lead to less
Ab affinity, the B cell dies.