Generation of antigen receptor diversity

Question 1

What are the five types of mammalian Ig heavy chains and their corresponding Igs

Answer 1

There are five types of mammalian Ig heavy chains denoted by Greek letters: α, δ, ε, γ and μ. These chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively.

Question 2

What are the two types of light chains

Answer 2

Kaaba and Gamma

Question 3

What is another word for hypervariable regions?

Answer 3

Complementary-determining regions (CDRs)

Question 4

What do CDRs determine?

Answer 4

Antigen specificity

Question 5

Do CDRs indirectly or directly interact with the antigen?

Answer 5

Directly

Question 6

How is the variable region generated?

Answer 6

V(D)J recombination reaction

Question 7

Junctional diversity increases in which hypervariable region and why

Answer 7

Junctional diversity increases the variability in the third hypervariable region, as its formed at the junction of the V and J gene segments.

Question 8

VJ recombination occurs in which chain

Answer 8

Light

Question 9

VDJ recombination occurs in which chain

Answer 9

Heavy

Question 10

How many possible combinations of VDJ segments are there?

Answer 10

1.9 x 10^6

Question 11

What fragments in the heavy chain of VDJ recombination join first?

Answer 11

D and J

Question 12

Do the V(D)J fragments attach with the C region at the start or end

Answer 12

End

Question 13

What proteins recognise recombinant signal sequences (RSS)

Answer 13

RAG proteins

Question 14

RSS

Answer 14

Recombinant signal sequences

Question 15

RAG1 and RAG2 are found on what human and mouse chromosomes

Answer 15

Found on chromosome 11p in humans and 2p in mice.

Question 16

RAG1 has what active site motif

Answer 16

DEE

Question 17

Where is the active region on the RAG-1 protein

Answer 17

C-terminal

Question 18

What amino acids does RAG1 act on

Answer 18

384-1008

Question 19

What amino acids does RAG2 act on

Answer 19

1-387

Question 20

Where is active region on RAG-2

Answer 20

N-terminal

Question 21

C-terminus on RAG-2 has what type of finger and what is it critical for

Answer 21

C-terminus has PHD (propyl hydroxylase domain) finger that is critical for chromatin binding

Question 22

What is the 12/23 rule in joining gene fragments

VDJ recombination

Answer 22

Combination can only occur between a 12-bp and a 23-bp spacer on RSSs

Question 23

Outline 4 steps of V(D)J recombination

Answer 23

RAG binding and nicking
Synapsis- OH groups
Hairpin formation and cleavage
Hairpin opening and joining + postcleavage complex

Question 24

RAG1 causes a nick on one strand, what group attacks the opposite strand. And what type of reaction is this, leading to what structure

Answer 24

3’OH
Transesterification
Hairpin at coding end, blunt ds break at signal ends

Question 25

How are coding joints processed

Answer 25

Ku70:Ku80 binds DNA ends, where then DNA-PK opens the hairpin and terminal deoxynucleotidyl transferase (TdT) processes the ends. Then DNA ligase, ligates the DNA ends.

Question 26

How are signal ends processed

Answer 26

Signal ends (circlular structure forms with NHEJ factors): Ku70:Ku80 binds DNA ends, and there are 5’ phosphorylated blunt ends. DNA ligase directly ligates the DNA ends through NHEJ.

Question 27

Diversity of human immunoglobulins are generated by joining different gene segments. Additional diversity is generated by processing the ________ ends, ______ to joining

Answer 27

Additional divertsity is generated by processing the coding ends PRIOR to joining.

Question 28

What is class switching? How does this relate to VDJ complex

Answer 28

Class switching is where an activated B cell change the Ab they produce, deletions of germline DNA results in religation of the VDJ complex to downstream heavy chain C region genes --> heavy chain switched.

Question 29

What is somatic hypermutation, in terms of Igs

Answer 29

Introduced point mutations into variable regions of Ig genes in B cells during germinal centre response

Question 30

What is the function of SHH (sonic hedgehog)

Answer 30

Survival signal preventing apoptosis in germinal centre B cells.

Question 31

What is gene conversion?

Answer 31

Gene conversion is a mechanism of antibody diversification where a non-reciprocal transfer of genetic information occurs between homologous DNA sequences, creating new variations in antibody genes

Question 32

Function of IgM

Answer 32

Activate complement

Question 33

Function of IgD

Answer 33

Developing tolerance?

Question 34

Function of IgG

Answer 34

Activate complement

Question 35

Where is IgA found

Answer 35

Secretions

Question 36

What is IgE involved in

Answer 36

Allergies

Question 37

Mature naive B cells express what Igs. How is differential expression achieved

Answer 37

Mature naïve B cells express IgM and IgG, differential expression is achieved through alternate splicing.

Question 38

Switching Ig isotopes to IgE, IgG, and IgA requires?

Answer 38

Class switch recombination

Question 39

What is the function of the AID-mediated cytidine deamination process

Answer 39

Generated nicks on both DNA strands leading to breaks in switch regions

Question 40

Outline process of class switching

Answer 40

DNA in variable or switch region Transcription produces local ssDNA In B cells, ssDNA is attacked by AID AID transition state Regeneration of AID and uridine produced Uracil-DNA-glycosylase (UNG) removes uracil to form apyrimidinic residue Apurinic/ apyrimidinic endonuclease (APE1) excises ribosome to form a single strand nick in DNA ssDNA nick formed DNA-PK and other repair proteins ac to initiate a ds break repair (DSBR) DSBR machinery joins the two switch regions and excises intervening sequences Selected constant region is now located adjacent to the VDJ region

Question 41

What is hypermutation triggered by?

Answer 41

Cytidine deaminase and uracil DNA-glycolase

Question 42

What cancers can appear from aberrant production of antigen receptors?

Answer 42

Lymphoid cancer

Question 43

Lymphoid cancers can have chromosome translocations involving antigen receptor loci ______

Answer 43

34

Question 44

NGS shows lymphoid cancers often have.........

Answer 44

structural variants involving RSSs at the breakpoint(s)

Question 45

What is cryptic recombination and 2 examples

Answer 45

Recombination with a cryptic RSS outside of the antigen receptor (RSS and non-consensus RSS). Examples are LMO2, SIL/SCL and TCR/IgH inversion (Ig heavy chain)

Question 46

What is end-donation. Give examples

Answer 46

Broken end on other chromosome aberrantly recombined into antigen receptor loci Examples: BCL-2/IgH – most common translocation in human cancer, accounting for most follicular lymphomas Mantle lymphoma: BCL1-IgH translocation

Question 47

What can the recombinant by-product do at an RSS

Answer 47

re-integrate

Question 48

What is cut and run? The breaks formed colocalised with those found in what cancer?

Answer 48

Cut-and-Run: The by-product stimulates RAG cutting at an RSS but itself remains uncut The breaks generated by cut-and-run: colocalsie with those found in acute lymphblastic leukaemia, map to cancer driver genes.

Question 49

Errors in class switching recombination lead to oncogene activation or tumour suppressor gene suppression

Answer 49

Oncogene activation

Question 50

Example of: Errors in class switch recombination lead to oncogene activation:

Answer 50

Translocation of the C-MYC gene to the IgH switch regions appears to be an error in CSR (AID-dependent translocation) Results in aberrant MYC activation and Burkitt’s lymphoma

Question 51

How can oncogenes be activated through AID

Answer 51

Mis-targeting of AID leads to oncogene activation through somatic hypermutation