GENETIC BASIS OF
GENETIC BASIS OF ANTIBODY DIVERSITY
Define the following terms: allelic exclusion, isotype switching,
affinity maturation, antibody repertoire, alternative RNA
splicing, recombination signal sequence.
Describe the genes that encode Ig Heavy and Light chains.
Describe the sequence of Ig gene rearrangement that occurs
during B-cell differentiation.
Discuss how diversity in antibody specificity is achieved.
Discuss the mechanisms of heavy chain class switching.
Calculate the number of possible Igs which can be produced
from given number of V, J, D, and C genes.
An antibody molecule is composed of two identical Ig heavy chains
(H) and two identical light chains (L), each with a variable region (V)
& constant region (C).
One of the important feature of the vertebrate immune system is its
ability to respond to an apparently limitless array of foreign
As immunoglobulin (Ig) sequence data accumulated,virtually every
antibody molecule studied was found to contain a unique amino acid
sequence in its variable region but only one of a limited number of
invariant sequences in its constant region.
The genetic basis for this combination of constancy and tremendous
variation in a single protein molecule lies in the organization of the
An Ab combining site is made up of one VL and one VH.
The specificity of any combining site is determined by its amino
There exist at least 106 unique combining sites .
Three families of Ig genes exist in mammals, one encoding HEAVY
chains, another KAPPA chains, and the third LAMBDA chains.
Each of these clusters contains one or more constant region genes
and a number of variable region gene segments.
The formation of a complete variable region of a light or heavy
chain requires the joining of two or three separate genetic elements
by a process of GENE REARRANGEMENT; a separate DNA
rearrangement in the heavy-chain complex is required for
Both germ-line and somatic events contribute to antibody
diversity, including COMBINATORIAL JOINING, SOMATIC
MUTATION and COMBINATORIALASSOCIATION.
GERM-LINE THEORY –
For every kappa-chain V-region there exists one unique
germ-line gene. A particular antibody-forming cell
selects one of these and expresses it in unmodified
SOMATIC THEORY –
Only a single germ-line gene exists for all kappa-chain
A particular antibody-forming cell expresses this gene
following a process of somatic mutation, which results in
each cell expressing a different version of this gene.
Dreyer and Bennett proposed the Two-Gene Model.
In 1965-Proposed that two separate genes encode a single
immunoglobulin heavy or light chain, one gene for the V
region (variable region) and the other for the C region
They suggested that these two genes must somehow come
together at the DNA level to form a continuous message that
can be transcribed and translated into a single Ig heavy or
Moreover, they proposed that hundreds or thousands of V-
region genes were carried in the germ line,whereas only
single copies of C-region class and subclass genes need
STRUCTURE AND EXPRESSION OF IMMUNOGLOBULIN GENES
Three families of immunoglobulin genes exist, each on a
kappa genes- chromosome 2
lambda genes- chromosome 22
heavy chain genes- chromosome 14
Each family consists of a series of V-regions
genetically linked to one or more C-regions.
STRUCTURE OF THE KAPPA CHAIN COMPLEX
This complex consists of a large number of V-region genes (about
55) genetically linked (by a long stretch of DNA) to a single copy of
the constant region gene.
An additional cluster of five short gene segments called J-segments
is located a few thousand base pairs upstream (5’ direction) of the C-
region gene, and each of these codes for the last 13 amino acids of
the variable region.
EXPRESSION OF KAPPA GENE:
The first step in expression of this gene is DNA REARRANGEMENT-
involving the joining of one V-region and one J-segment, each
chosen at random in any given B-cell.
The resulting structure have the DNA which was originally between
the selected V and J genes is cut out and lost in the form of a closed
V-region gene segments which happen to reside outside
this excised segment are retained, although they are no
longer relevant to expression.
This process is unique to immunoglobulin (and T-cell
The process of TRANSCRIPTION starts at the
beginning of the rearranged V-region and continues past
the end of the C-region, resulting in an immature mRNA.
The large intervening sequence (―intron‖) between the J-
segments and theC-region is removed by the process of
RNA SPLICING, resulting in the mature mRNA.
MOLECULAR BASIS OF KAPPA GENE EXPRESSION
Following DNA rearrangement:
Following RNA splicing:
Following peptide processing:
L V J C
V J C
J1 J2 J3J4 J5 CV1 V2 V3 . . .
V2 J3 CV1
V2 J3 C
V2 J3 C
transcription of mRNA
The structure and mechanism of expression of lambda chains and
heavy chains are similar to what we have just described for kappa
chains--all have J-SEGMENTS, all show DNA
REARRANGEMENT, TRANSCRIPTION, RNA
SPLICING, TRANSLATION and proteolytic cleavage of the
Heavy chain gene structure is somewhat more complex- there exists
an additional cluster of gene segments (known as “D”, for
“diversity”, segments) which each encodes four amino acids
between the V-region cluster and the J-segments.
DNA rearrangement for H-chains thus involves two events, joining
of a V with a D, and joining of the D with a J-segment.
Transcription and the other processes discussed above take place as
they do for kappa genes.
In each case, the end result is a polypeptide whose amino acid
sequence has been determined by three or four separate genetic
elements, and which is incorporated into the final immunoglobulin
ALTERNATE SPLICING IN B-CELLS
the simultaneous synthesis of IgM and IgD by a single B-cell.
This is the only example of a normal cell simultaneously producing
two different kinds of immunoglobulin.
The explanation derives from the fact that-mu (μ) and delta (δ)
constant region genes are adjacent to one another in the heavy chain
Using the same rearranged heavy chain V/D/J complex, a B-cell can
make two kinds of mRNA--it can transcribe from the V-region
through the end of the Cμ gene and make IgM, or it can transcribe
all the way through the Cδ gene and make IgD by splicing out the
Cμ region together with the intervening sequence during RNA
Two different mRNAs can thus be made from a single gene complex.
It should be emphasized that alternate splicing of RNA is a
mechanism used by many other genes to generate diverse protein
Dashed lines in RNA indicate an intervening
sequence removed during RNA processing
V J C CDL
SIMULTANEOUS SYNTHESIS OF IgM AND IgD IN B-CELLS
BY ALTERNATE RNA SPLICING
V JDL C
V JDL C
IgM and IgD synthesis in B-cells can occur simultaneously
The mu and delta chains are produced from the same
chromosome, and not from the two different allelic copies.
This is an extension of allelic exclusion is known as haplotype
In the choice of whether a secreted versus a membrane-
bound form of Ig is produced.
MECHANISMS FOR GENERATING ANTIBODY DIVERSITY
Multiple germ-line gene segments
Combinatorial V-(D)-J joining
P-region nucleotide addition (P-addition)
N-region nucleotide addition (N-addition)
Combinatorial association of light and heavy
MULTIPLE GERM-LINE GENE SEGMENTS
There are 51 VH, 25 D, 6 JH,40 V, 5 J, 31 V, and 4
J gene segments.
Many psuedogenes also influence.
Multiple germ-line V, D, and J gene segments
clearly do contribute to the diversity of the antigen-
binding sites in antibodies.
COMBINATORIAL V-(D)-J JOINING
The ability to create many different specificities by
making many different combinations of a small
number of gene segments.
Each V, D and J segments of DNA are flanked by
special sequences (RSS—recombination signal
sequences) of two sizes.
Single turn and double turn sequences (each turn
of DNA is 10 base pairs long).
Only single turn can combine with a double turn
Joining rule ensures that V segment joins only with
a J segment in the proper order.
Recombinases join segments together.
2.3 X 107
The enormous diversity generated by means of V,
D, and J combinations is further augmented by a
phenomenon called junctional flexibility.
Recombination involves both the joining of
recombination signal sequences to form a signal
joint and the joining of coding sequences to form a
joining of the coding sequences is often imprecise.
Cause addition/deletion of nucleotides in CDR.
Resulting in antibody diversity.
Joining of V and the J, or V, D and J segments
To form CODING JOINT is Imprecise
Addition and Deletion of Nucleotides
Diversity of the Hypervariable region 3 (CDR3)
P-REGION NUCLEOTIDE ADDITION (P-ADDITION),N-REGION
P-Addition:-Adds Diversity at Palindromic
During recombination some nucleotide bases are
cut from or add to the coding regions (p
Up to 15 or so randomly inserted nucleotide bases
are added at the cut sites of the V, D and J regions
By TdT (terminal deoxynucleotidyl transferase) a
unique enzyme found only in lymphocytes.
N NUCLEOTIDE ADDITION AT JOINING SEGMENTS: THE ADDITION OF
SOMATIC HYPERMUTATION ADDS EVEN
B cell multiplication introduces additional
opportunities for alterations to rearranged VJ or
These regions are extremely susceptible to
mutation compared to ―regular‖ DNA, about one
base in 600 is altered per two generations of
dividing (expanding) lymphocyte population
COMBINATION OF HEAVY AND LIGHT CHAINS ADDS FINAL
DIVERSITY OF VARIABLE REGION
8262 possible heavy chain combinations
320 light chain combinations
Over 2 million combinations
P and N nucleotide additions and subtractions multiply
this by 104
Possible combinations over 1010
LOCATION OF VARIABILITY OCCURS WITHIN CDR REGIONS OF V
DOMAINS (ANTIGEN BINDING SITES)
Is the conversion of an immunoglobulin from one isotype to another(e.g. IgG to
IgE) while retaining the same antigen specificity.
Switching is dependent on antigenic stimulation and is induced by
cytokines released by helper T cells and requires engagement of CD40L.
[e.g. IL-4 triggers switching from IgM to IgE or IgG4 (humans); IFN-γ triggers
switching from IgM to IgG2a (mice)].
Cyokines are thought to alter chromatin structure making switch sites
more accessible to recombinases for gene transcription.
Involves switch sites located in introns upstream of each CH segment
Switch sites consist of multiple copies of conserved repeat sequences
[(GAGCT)n GGGGGT)]; where n can vary from 3-7.
Class switching occurs usually in activated B cells (including memory
cells) and not in naïve B cells and involves heavy chain genes.
These cells (you will recall) already have rearranged VDJ genes at the
DNA level and were producing IgM and IgD.
CLASS SWITCHING AMONG CONSTANT REGIONS:
GENERATION OF IGG, IGA AND IGE WITH SAME ANTIGENIC
ISOTYPE SWITCHING CAN OCCUR BY:
Switch recombination (Deletion of
-primary mechanism of isotype
Alternative splicing of primary RNA
-Explains co-expression of multiple
isotypes by a single B cell.
SYNTHESIS, ASSEMBLY AND
REGULATION OF IG GENE TRANSCRIPTION
Each lymphocyte rearranged gene has regulatory
sequences that control gene expression
Promoters: initiation sites of RNA transcription
Enhancers: upstream of downstream that transcription
from the promoter sequence
Silencers: down-regulate transcription in germline cells
Gene rearrangement brings enhancer and promoter
regions close together and eliminates silencer regions
UNDERSTANDING OF IMMUNOGLOBULIN STRUCTURE AND
FORMATION HAS OPENED UP A NEW WORLD OF
Engineering mice with human immune systems
Generating chimeric and hybrid antibodies for clinical use
Abzymes: antibodies with enzyme capability