DNA recombination and the immune system

Question 1

………immune cells activate adaptive immunity

Answer 1

Innate

macrophages, granulocyte, natural killer cells

Question 2

site of development of B and T cells

Answer 2

B and T cells develop in the Bone-marrow and Thymus respectively
• Naïve B & T cells are present and surveilling the body for infection constantly
• There are approximately 1012 different B cells and 1012 different T cells
(i.e. with different receptors)
• Once activated the specific clone expands to make many copies of itself

Question 3

BCR (antibodies) and TCR see different antigenic determinants

Answer 3

Epitopes are regions of antigen recognized by the TCR and BCR
The epitopes recognized by T cells are often buried
the antigen
the epitopes recognized by the B cell are on top of the antigen

Question 4

Epitopes recognized by T cells

Answer 4

The epitopes recognized by T cells are often buried
the antigen must first be broken down into peptide fragments
the epitope peptide binds to a self molecule, an MHC molecule
the T-cell receptor binds to a complex of MHC molecule and epitope peptide

T-cell epitopes are peptides derived from antigens and recognized by the T-cell receptor (TCR) when bound to MHC molecules displayed on the cell surface of APCs

Question 5

BCRs (antibodies) and TCRs share common structural feature

Answer 5

The antigen binding site is in the variable portion of the receptors
• The antigen binding site is contributed to by two different polypeptide chains
• The constant portion of the receptors confers functional attributes unrelated to antigen specificity

Question 6

BCRs (antibodies) and TCRs have distinct structural features

Answer 6

• The BCR (antibody) consists of two heavy
and two light chains linked by di-sulphide bonds
• The TCR consists of one alpha and one
beta chain
• The BCR can be secreted as an antibody by the B cell after it has been fully activated
• The TCR is membrane bound and never
secreted

Question 7

Each lymphocyte chain has three hypervariable regions

Answer 7

• Each BCR and TCR is unique
• The antigen binding site has the most sequence variability
• These regions are known as hypervariable (HV) regions or complementarity determining regions
(CDR)
• There are three HV regions on each chain
• The most variable of the three HV regions is HV3 in each chain
(heavy, light, alpha, beta)

Question 8

Question: B and T cells perform different functions in the immune system.
Which of the following lymphocyte receptor features is responsible for the
neutralisation of extracellular pathogens?
A. T cells have an alpha and a beta chain that makes up the TCR
B. B cells have a BCR that can be secreted
C. T cells have a TCR that is membrane bound
D. B cells have a BCR with two heavy chains and two light chains

Answer 8

B. B cells have a BCR that can be secreted

Question 9

Difference between Heavy and light chain

Answer 9

Heavy chain contains V, J and D

light chain contains V and J only, no D (T cell alpha receptor is like the light chain)

Question 10

The number of different lymphocyte receptors is far greater than
the number of genes in the entire genome how?

Answer 10

B cell receptors (BCRs) and T cell receptors (TCRs) are
composed of polypeptide chains which come together to form receptors with defined specificity
The BCR and TCR genes encoding these polypeptide chains
are transcribed and translated just like the other ~3 x 104 genes encoding cellular components
The generation of lymphocyte receptor diversity
through somatic recombination

Question 11

Does genetic recombination occur in somatic cell?

Answer 11

NO
DNA recombination occurs during meiosis in the
formation of ‘germ cells’ or gametes
Does not occur in our somatic cells, whose DNA is
preserved in un-recombined form as the genetic blueprint for transcribing and translating the required products
Each cell in your body has the same DNA sequence
Lymphocytes are an exception to this rule. DNA
recombination (somatic recombination) does occur in
lymphocytes
Involves physically cutting out small regions of DNA and recombining these to create unique truncated sequences
The unique DNA sequences encode unique polypeptide chains that create unique lymphocyte receptors

Question 12

The DNA in each of our somatic cells is the same

EXCEPT IN

Answer 12

LYMPHOCYTES!!!
Expression of our genes in
MOST cells involves:
Transcription (mRNA) and
Translation into protein…..
But the blueprint (DNA) remains
unchanged

Question 13

The DNA sequence of the BCR heavy chain gene from

a B cell clone and a somatic cell differs

Answer 13

The somatic cells each carried the ‘blueprint’ DNA inherited from the parents
The DNA in each mature B cell clone had been rearranged with loss of a
large amount of the genetic information (black boxes)
Each B cell clone has a different rearrangement of the ‘blueprint’ DNA

Question 14

Recombination and expression of BCR heavy chain gene in two clones

Answer 14

Recombination first occurs between Diversity (D) and Joining (J) regions - 1 D and 1 J
Recombination next occurs between Variable (V) and DJ regions
The recombined gene is transcribed- primary RNA transcript
The VDJ complex is spliced onto the Constant (C) region RNA- messenger RNA
The mRNA is translated to produce the BCR heavy chain polypeptide

Question 15

combinatorial diversity

Answer 15

number of possible V(D)J combinations

Question 16

Question: How would you calculate the number of TCRs that could be generated if there are 45 Va, 50 Ja, 48 Vb, 2 Db, 12 Jb gene segments?
A. 45 + 50 + 48 + 2 + 12
B. 45 x 50 + 48 x 2 x 12
C. 45 x 50 x 48 x 2 x 12
D. 45 + 50 x 48 + 2 + 12

Answer 16

C. 45 x 50 x 48 x 2 x 12

Question 17

Hypervariable regions are encoded by

Answer 17

Variable (V) Diversity (D) and Joining (J) gene segments
Hypervariable regions 1 and 2 are encoded within V gene segments
• Hypervariable region 3 is encoded by a combination of V,D and J for
• BCR heavy chain
• TCR beta chain
• Hypervariable region 3 is encoded by a combination of V and J for
• BCR light chains
• TCR alpha chain

Question 18

Lymphocyte receptor diversity is contributed to by

three mechanisms - what are they

Answer 18

1. Recombination of lymphocyte receptor genes
2. Nucleotide deletion at junctions
3. Addition of nucleotides at junctions

Question 19

Somatic recombination occurs during

Answer 19

B and T cell development
B cells rearrange their BCR in the Bone marrow
T cells rearrange their TCR in the Thymus

Question 20

Recombination of the BCR and TCR genes involves co-ordinated activity of several enzymes

Answer 20

• Recombination activating gene (RAG) 1 and 2 enzymes
• Expressed only in developing B and T cells
• DNA repair enzymes
• Found in all cells and involved in DNA damage repair

Question 21

The mechanism of V(D)J rearrangement

Answer 21

RAG enzymes recognise specific Recombination Signal Sequences (RSS) in lymphocyte receptor genes to initiate recombination
• Sequences found
• 3' of each V gene segment
• 5' of each J gene segment
• Both sides of each D gene segment
• Consist of
• Conserved 7 nucleotide heptamer
• 12 or 23 non-conserved nucleotide spacer
• 9 nucleotide nonamer

Question 22

Recombination only occurs between RSS sites that have

Answer 22

12 bp and 23 bp spacers

A DH gene segment can be joined to a JH gene
segment
• A VH gene segment to a DH gene segment
• VH gene segments CANNOT be joined to
JH gene segments directly, as both VH and
JH gene segments are flanked by 23 bp spacers

Question 23

12/23 rule

Answer 23

Recombination only occurs between segments flanked by 12 bp spacer and 23 bp spacer
Ensures that recombination will not occur between only V elements (or only J elements)

Question 24

Question: The 12/23 bp spacer of the Recombination Signal Sequence (RSS) prevents the binding of which sequences?
A. TCR Db to Jb gene segments
B. TCR Va to Ja gene segments
C. BCR DH to JH gene segments
D. TCR Vb to Jb gene segments

Answer 24

Rag 1 and Rag 2 enzymes mediate the synapse of two RSS sites

with 12 bp and 23 bp spacers and cleave

Question 25

Rag 1 and Rag 2 function

Answer 25

Rag 1 and Rag 2 enzymes mediate the synapse of two RSS sites with 12 bp and 23 bp spacers and cleave
- Rag protein complexes binds to 12 and 23 bp spaced recombination signal sequence
- Binding together of RAG enzymes and bringing
together gene elements (creates a loop )
-Cleavage of DNA to create a hairpin structures at the end of immunoglobin gene segments
- other enzyme (ku70:ku80 and DNA-dependent protein kinases bind to the hair pin and cleave the RSS- hair pin cleaved at random
-DNA ligase IV and XRCC4 join the ends of the gene segments to form the coding joint

Question 26

Question: Somatic recombination in lymphocytes involves a number enzymatic
processes. Which of the following enzymes are uniquely active in lymphocytes?
A. RAG-1 and RAG-2
B. Ku70 and Ku80
C. DNA ligase IV
D. XRCC4

Answer 26

A. RAG-1 and RAG-2

Question 27

Junctional diversity is created by

Answer 27

the addition and subtraction of nucleotides to the coding joint
Terminal deoxynucleotidyl Transferase (TdT)
can add nucleotides not encoded
in the genome
DNA exonucleases can remove nucleotides

Question 28

The subtraction of nucleotides at the coding joint

Answer 28

Catalysed by DNA exonucleases, removes nucleotides

before the V (D) and J segments are joined

Question 29

Junctional diversity - introduces further diversity into the CDR3 region

Answer 29

Performed by enzymes
• RAG 1 and 2 to generate breaks
• DNA polymerase to “fill in” overhangs
• Exonuclease, and TdT (terminal deoxyribonucleotidyl transferase) direct the
subtraction or addition of nucleotides to ends - encoding novel amino acids
• Not always functional
• Addition of nucleotides can introduce frame shifts with stop codons or encode
molecules with different amino acid composition and thus structure
• Combinatorial diversity can generate ~1.6 x 106 different receptors
• Junctional diversity can increase this by a factor of ~3 x 107
• Totally the figure approaches ~4.8 x 1013 (48 trillion

Question 30

Question: Complementary Determining Region (CDR) - 3, also known as hypervariable regions
(HVR) - 3, of the antigen binding site of lymphocyte receptors is the most diverse region of the
receptor. The most comprehensive explanation for this diversity is that it is encoded by:
A. The variable (V) gene segments
B. Recombination of the V(D)J gene segments
C. Recombination of the V(D)J gene segments and the addition of bases at the junctions of the gene segments
D. Recombination of the V(D)J gene segments and the addition and subtraction of bases at the junctions of the
gene segments

Answer 30

D