8 - Generation of Diversity in B cell Ab receptors - Partridge

Question 1

Describe the 2 factors involved in recombination

Answer 1

Recognition signal sequences (RSSs), lymphocyte specific recombinases

Question 2

What makes up the RSS?

Answer 2

conserved heptameter (7bp) and nonamer (9bp) separated either by 12 or 23 random nucleotides

Question 3

Where are the RSSs found in the genome and overall what do they do?

Answer 3

RSSs are found directly adjacent to gene segments encoding V,D,J regions. they guide the rearrangement of these segements

Question 4

Draw a diagram illustrating the lambda, kappa and heavy chain genes and their RSSs

Answer 4

311 - 8

word

Question 5

What is the 12-23 base pair rule and why is this important?

Answer 5

a gene segment with a 12bp spacer will only recombine with a gene segment with a 23bp spacer. this ensures correct VDJ joining. eg V Lambda only joins with J lambda (not J Kappa). Also important because 12/23 corresponds to 1/2 turns of the DNA helix. therefore enzymes act on same side of DNA allowing them to facilitate the joining process

Question 6

What is the V(D)J recombinase responsible for?

Answer 6

somatic V region gene recombination

Question 7

What are the enzymes that make up this complex?

Answer 7

• DNA cleavage and repair enzymes eg DNA dependent protein kinase
• products of RAG1/2 genes (recombination activation genes). specialised endonuclease expressed in developing lymphocytes (important in only being expressed in developing kind)
• terminal deoxynucloetide transferase

Question 8

What does mutations in DNA-dependent protein kinase and the products of RAG1/2 genes result in?

Answer 8

scid. severe combined immunodeficiency. Ab/T cell responses are not efficient

Question 9

Draw a diagram highlighting the process of somatic recombination and explain this process

Answer 9

311 - 8 word

• RAG1/2 complex recognises and aligns the RSS adjacent to gene segments to be joined
• endonuclease activity of the RAG1/2 complex cleaves the DNA
• cleaved DNA repaired -> coding joint (V and j segments now next to each other) and the signal joint (intervening DNA is excised)

Question 10

State the 4 main ways of generating diversity in Abs / B cell receptors.

Answer 10

1. multiple copies of V region gene segments (Vn x Jn - light chain; Vn x Dn x Jn - heavy chain)
2. heavy x light chain combination (Vk x Jk OR VL x JL) + (VH x DH x JH)
3. imprecise recombination leading to junctional diversity.
4. somatic mutations of V regions following antigen activation

Question 11

Explain further imprecise recombination -> junctional diversity and somatic mutations of V regions.

Answer 11

imprecise recombination;
nucleotides are lost or added. variable addition of nucleotides @ junctions contributes to CDR3 diversity.
terminal deoxynucleotide transverse (TdT) randomly adds nucleotides to VDJ joins.

somatic mutation;
point mutation results in single bp changes. these base changes tend to be clustered in CDRs. this occurs when B cells undergo differentiation in response to antigen.

Question 12

outline the overall process of antigen independent B cell differentiation

Answer 12

1) heavy chain gene rearrangement. (D-J then V-DJ) to make u heavy chain.
2) light chain gene rearrangement (V-J) (k or L). -> express membrane IgM
3) selection against self molecules. unsure of the mechanism. isn’t perfect eg autoimmune diseases
4) passes into secondary lymphoid tissue where it meets antigen

Question 13

What immunoglobulins do virgin B cells predominantly express?

Answer 13

IgM OR IgM and IgG

Question 14

State the 2 forms of antigen dependent differentiation.

Answer 14

• somatic hypermutation

- class switch

Question 15

What is the main enzyme invovled in somatic hypermutation state its function?

Answer 15

AID; activation induced cytidine deaminase.

deaminates cytosine -> uracil

Question 16

Describe somatic hypermutation

Answer 16

• point mutations introduced into rearranged V regions. (1bp per 10^3 cell divisions. normally 1bp per 10^10 so increased mutation rate.)
• AID expressed in B cells in lymphoid tissue responding to antigen
• mutations tend to be clustered in CDRs in MATURE B cells. mutations can occur throughout V regions prior to this

Question 17

What are the functions of somatic hypermutation?

Answer 17

adding diversity but main function is affinity maturation.

Question 18

What does affinity maturation allow?

Answer 18

higher affinity receptors selected as immune response proceeds. survival of the fittest. when antigen becomes more scarce, ability of B cells to divide and differentiate depends on its ability of the Its to bind to antigen. those that dont bind antigen as tightly will not divide.

Question 19

What is class switching?

Answer 19

when the recombined V region (specific to antigen) associates with different C regions. Antigen specificity retained but now has different localisation/effector functions eg if we have parasitic infection, binding to antigen can still occur but if recombined with the C region of IgE then we can activate mast cells.

Question 20

What are the 2 things that class switching and somatic hypermutation is dependent upon?

Answer 20

AID and T cell help

Question 21

Draw a diagram of the arrangement of the heavy chain gene locus including the VDJ region and the C regions

Answer 21

311 - 8 word

Question 22

Draw a diagram showing how class switching occurs

Answer 22

311 - 8

Question 23

When does class switching occur?

Answer 23

once the B cell is responding to antigen in 2ndry lymphoid tissue

Question 24

Which C region does NOT have an S region before it?

Answer 24

IgD / delta

Question 25

how is this recombination different to the recombination we see at VDJ regions?

Answer 25

somatic recombination we see at VDJ gene fragments involve the RSSs. here, it occurs at switch regions and class switching initiated by AID acting at these switch regions.

Question 26

what are the 2 results of class switching by DNA recombination @ switch regions?

Answer 26

• intervening DNA looped out/excised out

- irreversible

Question 27

What does AID do specifically ?

Answer 27

deaminates a cytidine to uracil

Question 28

when is AID activated and how does it give rise to mutations?

Answer 28

AID activated when B cells responding to antigen (activated B cells). active on ssDNA.
gives rise to mutations following error-prone repair of the mutated DNA. eg base excision repair, mismatch repair.

Question 29

what are the 2 results of these mutations (as a result of AID activity)?

Answer 29

somatic hypermutation; Ab with higher affinity to antigen are selected for. 
class switching; AID particularly active in switch regions -> ss nicks eventually giving rise to ds nicks -> class switching

Question 30

What are the S regions composed of?

Answer 30

G rich tandem repeats near C regions

Question 31

What is associated with mutations in AID?

Answer 31

immunodeficiency. cannot give rise to Abs that have undergone somatic hypermutation or class switching

Question 32

Why is it important that AID is only expressed in activated B lymphocytes?

Answer 32

regulation needs to be tightly controlled. cannot be mutating DNA frequently.

Question 33

Which cancers have been shown to be caused by incorrect AID activation

Answer 33

gastric cancer. Helicobacter pylori shown to induce aberrant AID expression -> stomach ulcers/gastric cancer

Question 34

how can a B cell express 2 Ab classes on its surface?

Answer 34

initial, long primary transcript for the B cell immunoglobulins contain both IgM/IgD genes. differential transcript processing and splicing gives rise to 2 types of transcripts, both of which expressed on surface. this process is reversible.

Question 35

when does this co expression process occur?

Draw a diagram to help explain your answer

Answer 35

following VDJ recombination but prior to class switching

Question 36

how do we get the production of surface and secretory forms of an immunoglobulin from the same heavy chain gene? draw a diagram to help

Answer 36

differential processing of primary transcript determines whether cell makes secretory/surface form

Question 37

When does the cell start to produce more secretory Igs?

Answer 37

when B cell activated and responds to antigen

Question 38

describe the composition in terms of hydrophobic/hydrophillic of the secretory/surface parts of the immunoglobulin

Answer 38

Secretory; hydrophilic

Surface/membrane; hydrophobic