One of the important parts in the study of Immunology.I prepared it for the sake of a seminar series competition conducted in my university. Now I thought of sharing it with others.

1. ORGANIZATION AND
EXPRESSION OF
IMMUNOGLOBULIN
GENES
By,
Devika.R
M Sc Student, Dept of
Biotechnology
Gulbarga University,
Karnataka

2. Structure of Immunoglobulin

3. ORGANIZATION AND EXPRESSION OF
IMMUNOGLOBULIN GENES

4. GENETIC MODEL COMPATIBLE WITH
IMMUNOGLOBULIN STRUCTURE
Properties of Antibodies
 Vast diversity of antibody specificities.
 Presence in immunoglobulin heavy and light
chains of a variable region at the amino terminal
end and a constant region at the carboxy terminal
end.
 The existence of isotypes with the same antigen
specificities, which result from the association of a
given variable region with different heavy chain
constant region.

5. GERM LINE AND SOMATIC VARIATION MODELS
Two different sets of theories emerged to explain
tremendous diversity of antibody structure.
Germ line
• The genome contributed by the germ cells, eggs
and sperm contains a large repertoire of Ig genes.
Somatic variation
• The genome contains a relatively small no: of Ig
genes from which a large no: of antibody
specificities are generated in the somatic cells by
mutation and recombination.

6. How could stability be maintained in the constant (C)
region while some kind of diversifying mechanism
generated the variable (V) region?
TWO GENE MODEL OF DREYER AND BENNETT
In 1965, W. Dreyer and J. Bennett suggested 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 (constant
region).
These two genes must come together at the DNA
level to form a continuous message that can be
transcribed and translated into a single Ig heavy or
light chain.

7. TONEGAWA’S EXPERIMENT
 In 1976, S. Tonegawa and N. Hozumi found that
separate genes encode the V and C regions of
Immunoglobulins and that the genes are
rearranged in the course of B-cell differentiation.
 Tonegawa was awarded Nobel prize for this work
in 1987.

9. MULTIGENE ORGANIZATION OF
IMMUNOGLOBULIN GENES
 Germ-line DNA contains several coding sequences,
called gene segments, separated by non-coding
regions.
Gene segments are rearranged during B cell
maturation to form functional Ig genes.
 Each multigene family has distinct features.
The κ and λ light chain families contain V, J and C
segments; the rearranged VJ segments encode the
variable region of the light chains and the C gene
segments encode the constant region.

10.  The heavy-chain family contains V, D, J, and
C gene segments; the rearranged VDJ gene
segments encode the variable region of the
heavy chain.
Each V gene segment is preceded at its 5’ end
by a signal or leader (L) peptide that guides the
heavy or light chain through the endoplasmic
reticulum.
Signal peptide is cleaved from the nascent
light and heavy chains before assembly of the
final Ig molecule.

11. λ CHAIN MULTIGENE FAMILY
Functional λ variable-region gene contains two coding
segments – a 5’ V segment and a 3’ J segment – which
are separated by a non-coding DNA sequence in
unrearranged germ-line DNA.
 J for joining ( 39 bp gene segment).
 In mouse, λ multigene family contains 3 Vλ , 4 Jλ and
4 Cλ gene segments.
 Jλ 4 is a pseudogene or a defective gene that is
incapable of coding proteins, denoted by ψ.
 λ chain have 3 subtypes (λ 1, λ 2 and λ3).
 In humans, there are 31 functional Vλ , 4 Jλ and 7 Cλ
gene segments. It also contains many pseudogenes.

12. κ CHAIN MULTIGENE FAMILY
 In mouse, κ chain multigene family contains
approximately 85 Vκ , 5 Jκ (one pseudogene) and 1 Cκ
gene segment.
 Vκ and Cκ encode variable region of the κ light chain
and the Jκ gene segment encode the constant region.
 There are no subtypes of κ light chains.
 In humans, approximately 40 Vκ , 5 Jκ and 1 Cκ gene
segments are present.

14. HEAVY CHAIN MULTIGENE FAMILY
 Leroy Hood proposed the existence of an additional
gene segment in the heavy chain variable region.
 It was designated D for diversity because of its
contribution to the generation of antibody diversity.
 The heavy chain multigene family on human
chromosome 14 contain 51 VH, 27 DH , 6 JH and a
series of CH gene segments.
 The CH gene segments consist of coding exons and
non-coding introns.
 In humans and mice, the CH gene segments are
arranged sequentially in the order Cµ , Cδ ,Cγ ,Cε , Cα.

16. VARIABLE REGION GENE
REARRANGEMENTS
 Variable –region gene rearrangements occur in an
ordered sequence during B-cell maturation in the
bone marrow.
 The heavy chain variable -region rearrange first, then
light chain.
 Process of variable –region gene rearrangement
produces mature, immunocompetent B cells which
produce antibody with a binding site encoded by the
particular sequence of its rearranged V genes.
Rearrangements of the heavy chain constant region
genes will generate further changes in the isotype
expressed by a cell.

17. V-J REARRANGEMENTS IN LIGHT CHAIN DNA
 Expression of both κ and λ light chains requires
rearrangement of the variable-region V and J gene
segments.
 In humans, any of the functional Vλ genes can combine
with any of the four functional Cλ- Jλ combinations.
 In mouse, things are slightly more complicated.
 In human or mouse κ light chain DNA, any one of the Vκ
gene segments can be joined with any one of the functional
Jκ gene segments.
 Rearranged genes have a short leader (L) exon, a non-
coding sequence (intron), a joined VJ gene segment, a
second intron, and the constant region in order from 5’ to
3’ end.

19. V-D-J REARRANGEMENTS IN HEAVY CHAIN DNA
This process requires two separate rearrangement
events within the variable region.
 DH gene segment first joins to a JH segment; the
resulting DHJH segment joins a VH segment to generate
a VH DHJH unit that encodes the entire variable region.
Rearrangement produces a short L exon, an intron, a
joined VDJ segment, another intron and a series of C
gene segments.

21. MECHANISM OF VARIABLE REGION DNA
REARRANGEMENTS
Recombination signal sequences direct recombination

22. RSSs is found flanking each germ line V,D and J gene segment.
One RSS is located 3’ to each V, 5’ to each J and on both sides
of each D gene segments.
Functions as signals for the recombination process that
rearranges genes.
Each RSS contains a conserved palindromic heptamer and a
conserved AT-rich nonamer separated by 12- and 23- bp
sequences.
12 bp-One turn RSS and 23 bp-two turn RSS of DNA helix.
Signal sequences having one turn spacer can join only with RSS
having two turn spacer (one turn/two turn joining rule).

23. ENZYMATIC JOINING OF GENE SEGMENTS
 V-D-J recombination takes place at the junctions
between RSSs and coding sequences is catalyzed by
enzymes collectively called V(D)J recombinase.
 In 1990, David Schatz, Marjorie Oettinger and David
Baltimore first reported the identification of two
recombination-activating genes, RAG-1 and RAG-2.
 The RAG-1 and RAG-2 proteins and the enzyme
terminal deoxynucleotidal transferase (TdT) are
the only lymphoid specific gene products that had
been shown to be involved in V-(D)-J rearrangement.

25. PRODUCTIVE AND NON-PRODUCTIVE
REARRANGEMENTS
 Imprecise joining of rearranged segments may result
in out of phase joining and the triplet reading frame
for translation is not preserved.
 In such a non-productive rearrangement, the
resulting VJ or VDJ unit will contain numerous stop
codons which interrupt translation.
 When gene segments are joined in phase, the reading
frame is maintained.
 In such a productive rearrangement , the resulting
VJ or VDJ unit can be translated in its entirety, yielding
a complete antibody.

26. ALLELIC EXCLUSION
 A B cell is diploid, it
expresses the rearranged
heavy chain genes from
only one chromosome.
 The process by which this
is accomplished is called
allelic exclusion.
 It ensures that functional
B cells never contain more
than one VHDH JH and one
VLJL unit.
 It is essential for the
antigenic specificity of the
B cell.

27. GENERATION OF ANTIBODY DIVERSITY
Seven means of antibody diversification have been
identified in mice and humans.
1. Multiple germ-line gene segments
2. Combinatorial V-(D)-J joining
3. Junctional flexibility
4. P-region nucleotide addition
5. N-region nucleotide addition
6. Somatic hypermutation
7. Combinatorial association of light and heavy chains

28. 1.MULTIPLE GERM LINE 2.COMBINATORIAL V-D-J
GENE SEGMENTS JOINING
 In human germ line DNA, 51
VH, 27 D, 6 JH, 40 Vκ , 5 Jκ , 31
Vλ and 4 Jλ segments are
present.
 In mouse, about 85 Vκ , 134
VH ,4 functional JH , 4
functional Jκ ,3 functional Jλ
and an estimated 13 DH but
only 3 Vλ gene segments are
present.
They contribute to the
antigen-binding sites in
antibodies.
 In humans, the ability of any
of the 51 VH gene segments to
combine with any of the 27
DH segments and any of the 6
JH segments allows a
considerable amount of
heavy-chain gene diversity to
be generated (51276=8262
possible combinations).
 40 Vκ5 Jκ=200 and 30Vλ
4 Jλ=120 possible
combinations.
 Total antibody combining
diversity in human to well
over 1010 .

29. 3.JUNCTIONAL FLEXIBILITY
 Increases the enormous diversity generated by means
of V, D, and J combinations.
Can lead to many nonproductive rearrangements, but
it also generates several productive combinations that
encode alternative amino acids at each coding joint,
thereby increasing antibody diversity.

30. 4.P-ADDITION 5.N-ADDITION

31. 6.SOMATIC HYPERMUTATION
 Results in additional antibody diversity that is generated in
rearranged variable-region gene units.
 Also causes individual nucleotides in VJ or VDJ units to be
replaced with alternatives, thus possibly altering the
specificity of the encoded immunoglobulins.
 Usually occurs within germinal centers
 Targeted to rearranged V-regions located within a DNA
sequence containing about 1500 nucleotides, which
includes the whole of the VJ or VDJ segment.
 Largely random
 Affinity maturation: following exposure to antigen, those
B cells with higher affinity receptors will be selected for
survival because of their greater ability to bind to the
antigen this takes place in germinal centers.

32. 7. ASSOCIATION OF HEAVY AND LIGHT
CHAINS
# Combinational association of H and L chains also can
generate antibody diversity.
# In humans, potential no: of heavy and light chain
combinations is 2,644,240 (8262 heavy chain 320
light chain).
# This is higher than the actually generated amount
because not all VH , D or VL gene segments are used in
the same frequency.

33. CLASS SWITCHING AMONG CONSTANT
REGION GENES
Class Switching-VHDH JH unit combine
with any CH gene segment. Also called
isotype switching.
Involves DNA flanking sequences called
switch regions(2 to 10 kb). Switch
regions composed of short
repeats(GAGCT and TGGGG).
Cytokines(Intercellular regulatory proteins)
act as switch factors. Plays major role in
determining the Ig class expressed as a
result of switching.

35. EXPRESSION OF IMMUNOGLOBULIN GENES
Processing of primary transcript to
remove intervening sequence including
non coding introns and J gene
segments not lost during V-(D)-J
rearrangement.
Remaining exons are connected by a
process called RNA splicing.
Final mRNA product is then exported
from nucleus and then translated by
ribosomes into complete H or L chains.

36. DIFFERENTIAL RNA PROCESSING OF HEAVY CHAIN
PRIMARY TRANSCRIPTS
 Immunoglobulin can exist in either membrane-
bound or secreted form.
 Processing can yield different mRNA’s
This processing explains the production of secreted or
membrane bound forms of a particular
immunoglobulin and the simultaneous expression of
IgM and IgD by a single B cell.

37. 1.EXPRESSION OF MEMBRANE OR SECRETED
IMMUNOGLOBULIN
o Difference depends on differential processing of a
common primary transcript.
o Mature naive B cells produce only membrane-bound
antibody and differentiated plasma cells produce
secreted antibodies.
o The secreted form has a hydrophilic sequence of
about 20 amino acids in the C terminal domain; This is
replaced by a sequence of about 40 amino acids
containing a hydrophilic segment that extends outside
the cell, a hydrophobic transmembrane segment and a
short hydrophilic segment at C terminal that extends
into cytoplasm.

39. 2.SIMULTANEOUS EXPRESSION OF IgM AND IgD
 Controlled by differential RNA processing .
If the heavy chain transcript is cleaved and
polyadenylated at site 2 after the Cµ exons, then the
mRNA will encode the membrane form of the u heavy
chain.
If polyadenylation is instead further downstream at
site 4, after the Cδ exons, then RNA splicing will
remove the intervening Cµ exons and produce mRNA
encoding the membrane form of the δ heavy chain.

41. SYNTHESIS, ASSEMBLY, AND SECRETION OF
IMMUNOGLOBULIN

42. REGULATION OF Ig-GENE TRANSCRIPTION
Three regulatory sequences in DNA regulate transcription of
immunoglobulin genes:
1. Promoters: promote initiation of RNA transcription in a
specific direction. Located about 200 bp upstream from
transcription initiation site.
2. Enhancers: nucleotide sequences located some distance
upstream or downstream from a gene that activate transcription
from the promoter sequence in an orientation-independent
manner.
3. Silencers: nucleotide sequences that down-regulate
transcription, operating in both directions over a distance.
Each VH and VL gene segment has a promoter located just
upstream from the leader sequence.
These promoters contain a TATA Box, which serves as the site for
the binding of no: of proteins that initiates transcription.

43. EFFECT OF DNA REARRANGEMENT INHIBITION OF Ig GENE
ON TRANSCRIPTION EXPRESSION IN T-CELLS
Rate of transcription of a
rearranged VLJL or VH DH JH
unit is as much as 10,000X
the rate of transcription of
unrearranged VL or VH
segments.
Oncogenes: genes that
promote cellular
proliferation or prohibit cell
death. These can often
translocate to the
immunoglobulin heavy or
light chain loci.
Expression of these genes is
elevated significantly under
the influence of an Ig
enhancer.
T-cell receptor (TCR)
undergoes V-(D)-J
rearrangement to generate
functional TCR genes.
Occurs by the similar
recombination mechanism
of Ig .
Complete Ig gene
rearrangement of H and L
chains occurs only in B cells
and complete TCR-gene
rearrangement is limited to
T cells.

44. ANTIBODY GENES AND ANTIBODY
ENGINEERING
Possible to design and construct genes that encode
immunoglobulin molecules in which the variable regions
come from one species and the constant regions come from
another.
A. CHIMERIC AND HYBRID MONOCLONAL ANTIBODIES
• To make an antibody one needs to clone recombinant DNA
containing the promoter, leader, and variable-region sequences
from a mouse antibody gene and the constant-region exons from
a human antibody gene.
• The antibody encoded by such a recombinant gene is a mouse-
human chimera, also known as humanized antibody.

45. • Its antigenic specificity is determined by the variable region
and is derived from the mouse DNA.
•Its isotype is determined by the constant region and is derived
from the human DNA.
•Heteroconjugates (or bispecific antibodies): hybrids of two
different antibody molecules.
•Constructed by chemically crosslinking two different
antibodies or synthesized in hybridomas consisting of two
different monoclonal-antibody-producing cell lines that have
been fused.

47. B. MONOCLONAL ANTIBODIES CONSTRUCTED
FROM IG-GENE LIBRARIES
Use of PCR to amplify the DNA that encodes antibody
heavy chain and light chain Fab fragments from
hybridoma cells or plasma cells.
 A promoter region and EcoRI restriction site are added to
amplified sequence and the resulting constructs are
inserted into bacteriophage λ , yielding separate H and L
chain libraries.
 Random joining of H and L chains yield numerous novel
H-L constructs.
This procedure generates a diversity of antibody
specificities; clones containing these random
combinations of H + L chains can be rapidly screened for
those secreting antibody to a particular antigen.

48. MICE HAVE BEEN ENGINEERED WITH HUMAN
IMMUNOGLOBULIN LOCI
One can knockout the heavy and light chain
immunoglobulin loci in mouse embryonic stem cells
and introduce very large DNA sequences containing
human heavy and light chain gene segments.
Advantage of this process: completely human
antibodies are made in cells of the mouse B-cell
lineage, from which antibody-secreting hybridomas
are readily derived by cell fusion.
Therefore, this process offers a solution to the problem
of producing human monoclonal antibody of any
specificity desired.

50. SUMMARY
Ig light and heavy chains are encoded
by3 separate multigene families.
• Functional light and heavy chains are generated by random
rearrangement of the variable –region gene segments.
V(D)J joining is catalyzed by the
recombinase activating genes RAG-1 and
RAG-2 and other enzymes and proteins.
• Joining of segments is directed by RSS.
Ig gene rearrangements occur in
sequential order.
• Heavy chain rearrangements first, followed by
light chain rearrangements.

51. Major sources of antibody diversity are;
• Random joining of multiple V,J and D gene segments, junctional
flexibility, somatic mutation
• Random association of heavy and light chains, P addition and N addition.
Class switching results in expression of different classes of
antibody (Ig G, Ig A, Ig E) with the same antigen
specificity.
Differential RNA processing of the Ig heavy chain primary
transcript generates,
• Membrane bound antibody in mature B cells, secreted antibody in plasma
cells.
• And the simultaneous expression of Ig M and Ig D by mature cells.
It is possible to engineer antibodies for research and therapy
which includes chimeric antibodies, bacteriophage-based
combinatorial libraries of Ig genes and transplantation of
human Ig loci into mice.

52. REFERENCE
1. IMMUNOLOGY-Janis Kuby, 5th edition
2. ESSENTIALS IN IMMUNOLOGY-Lecture by
Prof. Anjali.M.Karande, IIS, Bangalore

53. THANK OU