Lecture 5 - Somatic Recombination

Question 1

Define the 4 Methods by which Receptor Diversity is generated, including in which cell types these occur

Answer 1

• Somatic Recombination (B/T-cells)
• Junctional Diversity (B/T-cells)
• Somatic Hypermutation (Only B-cells)
• Isotype Switching (Only B-cells)

Question 2

How is the Light Chain V-domain Constructed?

(2 Points)

Answer 2

• Light Chain V-domain is encoded in two gene segments: V gene segments and J/Joining gene segments
• V and J gene segments are joined by somatic recombination to produce continous exon encoding the full V domain

Question 3

How is the Heavy Chain V-domain Constructed?

(2 Points)

Answer 3

• Heavy Chain V Domain is encoded by three gene segments: V(H) segment, Joining/J(H) segment and the Diversity/D(H) segment
• First the D gene segment is joint to a J segment, then the V Segment is joined to the DJ segment to produce continuous exon encoding full heavy chain V domain

Question 4

How is complete heavy/light chain mRNA produced?

Answer 4

Joining of V region RNA and C region RNA via RNA Splicing

Question 5

How is DNA Rearangement Guided?

What is the Structure of this Sequence?

(Part 1 - 1 Point) (Part 2 - 3 Points)

Answer 5

• Guided by Recombination Signal Sequences (RSS) adjacent to site where recombination occurs
• RSS - consists of:
  • Heptamer
  • Spacer (12/23 Nucleotides)
  • Nonamer

Question 6

Why is Spacer Length Important?

(2 Points)

Answer 6

• Corresponds with One/Two turns of DNA double helix and brings heptamer and Nonomer to same face of helix
• This allows conserved regions of RSS to be bound by V(D)J Recombinase, the protein which catalyses recombination

Question 7

State the Subunits of VDJ Recombinase responsible for recognising/binding RSS?

Answer 7

RAG-1 and RAG-2 (Recombination Activating Genes)

Question 8

What is the 12/23 Rule?

Answer 8

Only a Gene Segment flanked by an RSS with a 12bp spacer can be joined to one with a flanked by an RSS with a 23bp spacer

Question 9

When Does Somatic Recombination Occur?

Answer 9

During Initial Development in Primary Lymphoid Organs (i.e., Bone Marrow, Thymus)

Question 10

Why Must the Initial Signal Joint be further processed into a precise signal joint?

Answer 10

To inactivate it in order to prevent its 5’ phosphorylated ends from integrating into other sites in genome

Question 11

How is Junctional Diversity Generated?

Answer 11

Random insertion of Nucleotides by TdT following opening of DNA hairpins

Question 12

Define Somatic Hypermutation in terms of:
(i) What it is?
(ii) How it works?
(iii) Where it occurs?
(iv) When it occurs?

Answer 12

(i) Additional Mechanism which Generates Diversity in V regions of B-cells only
(ii) Point Mutations occurs at increased rate in variable regions of antibody encoding genes, leading to alterations in affinity (affinity maturation)
(iii) Takes place in rearranged V regions in peripheral lymphoid organs
(iv) In response to antigen recognition and signals from effector cells

Question 13

Where is Junctional Diversity Generated in TCR Gene Rearrangement?

(2 Points)

Answer 13

• Between V, D and J segments of rearranged TCR B gene
• Between V and J segments of rearranged TCR a gene

Question 14

How is TCR Gene Rearrangement Guided?

Answer 14

TCR gene segments are flanked by RSS Homologous to those flanking Ig gene segments

Question 15

How is Isotype Switching Guided?

(2 Points)

Answer 15

• Guided by Repetitive sequences in the introns of C genes known as switch regions
• These lie upstream of each of the C genes for every heavy chain isotype (exception - delta is co-expressed with mu gene)

Question 16

Compare Isotype Switching to Somatic Recombination

(4 Points)

Answer 16

• Isotype Switching - always productive
• Different Recombination signals and enzymes involved
• Isotype Switching occurs post-antigen stimulation, whilst Somatic Recombination occurs during B-cell development in bone marrow
• Isotype Switching is not a random process, being regulated by external signals (e.g., Cytokines)