Boyes V(D)J Flashcards
(77 cards)
1
Q
Where does VDJ recombination occur?
A
Heavy chain in proB cells
Light chain in preB cells
2
Q
What size is each domain of an antibody?
A
110residues
3
Q
How many polypeptides is an antibody made up of?
A
4
4
Q
How many types of light chain constant region are there?
A
2- lamda and kappa
5
Q
How many types of antibodies are there?
A
5x10^13
6
Q
Which cells can rearrange the DNA?
A
B cells
7
Q
How many combinations of segments make up a heavy chain?
A
40V
25D
6J
=6000
8
Q
How many kappa variable segments are there?
A
40
9
Q
How many kappa Joining segments are there?
A
5
10
Q
How many lamda variable segments are there?
A
30
11
Q
How many lamda joining segments are there?
A
4
12
Q
How many combinations can VDJ create?
A
1.9x10^6
13
Q
Which proteins bind RSS?
A
RAG proteins
14
Q
What are the 2 types of RSS?
A
23bp or 12bp
15
Q
What is the sequence of 23bp RSS?
A
heptamer-spacer-nonamer
16
Q
What is the RSS sequence of kappa?
A
V23- 12J
17
Q
What is the RSS sequence of lamda?
A
V12-23J
18
Q
What is the RSS sequence of heavy chain?
A
V23-12D12-23J
19
Q
What is the sequence of the 12bp RSS?
A
nonamer-spacer-heptamer
20
Q
Is the RSS spacer sequence conserved?
A
No
21
Q
What is the structure of RAG1?
A
1040 residues,
C terminal active
DDE motif
binds nonamer
22
Q
What is the structure of RAG2?
A
527 residues
N terminal active
C terminal PHD binds 11p chromosome
binds heptamer
23
Q
Do RAGs have introns?
A
No
24
Q
What is the method of VDJ recombination?
A
RAGs bind RSS making synaptic complex
RAG1 produces a single strand nick
RAG2 transesterification produces a dsDNA break
NHEJC excise fragment
25
What is the structure of the heavy chain?
V-D-J-intron-C1-intron-Hinge-intron-c2-intron-c3
26
How far apart can RSS be?
20kb- 1Mb
27
What does RSS in the same direction cause?
Inversion
28
How is junctional diversity added?
```
Ku70:Ku80 binds ends
DNA-PK:Artemis opens ends assymetrically
P nucleotides added to fill in hairpin producing palindrome
TdT adds N nucleotides to heavy chain
exonuclease removed unpaired bases
DNA ligase 4 ligates ends
```
29
Where is junctional diversity produced?
Hypervariable region 3
30
When is junctional diversity produced?
light chain: pre-B cell stage
| Heavy chain: pro0B stage
31
How much variation is produced by junctional diversity?
3x10^7
32
Which constant chain do naive B cells express?
M or D immediately downstream
33
How are antibodies targetted to membranes or for secretion?
25 C terminal residues
34
Which antibody modifications are reversible?
Alternative splicing of IgM/D and membrane/secreted
35
How is affinity of IgM and IgA raised?
J protein binds C chain to make pentamers or dimers
36
What are the 5 isotypes of heavy chain?
M, D, E, A, G
37
Which heavy chain cannot be produced by class switching?
M
38
When does class switching occur?
In mature T cells that have been recruited to germinal centres in lymph nodes and recognising antigen.
39
What are switch regions?
1-10kb long G rich upstream regions
40
How does class switch recombination occur?
```
AID binds transcribed ssDNA
Zn mediated C deamination
UNG removes U base
APE1 removes backbone to create a nick
Lots of nicks, some randomly align to create dsDNA breaks
NHEJC joins ends to remove fragment
```
41
Can multiple class switches occur?
Yes but must be downstream each time
42
What does somatic hypermutation do?
Fine tunes affinity through 3 hypervariable sites
43
What are the 3 mechanisms of somatic hypermutation?
AID produces G=U mismatch
1) replication produces T in half daughter cells
2) UNG removes base, lesion bypass randomly inserts base
3) MMR elements insert A-T mutations
44
Why does AID not produce dsDNA breaks in somatic hypermutation?
Lower concentration
45
What are the 4 methods of adding diversity to the variable region?
V(D)J (1.9x10^6)
Junctional (3x10^7)
Combinatorial
Somatic Hypermutation
46
What are the structural differences in T cell antibodies?
Only one receptor,
made of 2 chains alpha and beta
only one variable region
47
How is diversity added to T cell antibodies?
No somatic hypermutation, more J segments
48
What type of T cell antibodies are produced in the gut?
gamma and delta
49
How are antibodies held in membranes?
complexes using 25residue C terminal motifs
50
Where are antigens presented?
APC/dendritic cells with MHC2 for bacterial peptides or MHC1 for viral peptides
51
How do T cells recognise antigens?
```
MHC class 2 (+bacteria) recognised CD4 complexed antibodies on T helper cells
MHC class 1(+virus) recognised by CD8 complexes on cytotoxic T cells
```
52
How is V(D)J regulated?
Limited to G1 phase by RAG2 pT490 degradation
RAGs only expressed in B and T cells
Epigenetics exclude RSS in one allele
53
What phase is V(D)J recombination limited to?
G1
54
How is V(D)J initiated?
Transcription removes H2A/B dimers to expose RSS in 35-40bp for ~6 minutes.
55
What does transcription of antigen receptor loci cause?
H3K4me3 which binds RAG2 PHD
| Acetylation opens chromatin structure and recruits remodelling complexes through bromodomains
56
What are the characteristics of the enhancers of antigen receptor loci?
Tissue, stage and locus specific
57
How has it been proved that transcription is essential for V(D)J?
Long sterile transcripts
Insertion of terminators downstream of J61 only allows transcription downstream of J49 promoter
Enhancer KO mice do not have V(D)J
58
How does allelic exclusion occur in V(D)J?
Only one enhancer and only one allele in active centre of nucleus
59
How has allelic exclusion been proved?
2 enhancers upregulates expression
60
What type of disease does errors cause?
50% of all lymphiod cancers
61
What is the main form of error during V(D)J?
Chromosome translocation during dsDNA breaks
62
How can chromosome translocations occur?
```
cryptic RSSs
End donation
RAG mediated transposition
ESC reintegration
Cut and run
```
63
What size is the ESC?
20kb-1mb
64
How many mutations does RAG mediated transposition cause?
1/44,000 recombination events
65
Where has RAG mediated transposition occured?
THRalpha/HPT locus
| IN VITRO
66
What protects against RAG mediated transposition in vivo?
The C regulatory domain of RAG
67
Where have cryptic RSSs been proved?
Sequencing of LM02 from chromosome 11 into T cell alpha locus of chromosome 14
68
Where have cryptic RSSs been found?
LM02/alpha
SIL/SCL
TCR/IgH
69
How do broken ends of chromosomes get incorporated?
RAG recognition of unusual hairpin structures
70
What are examples of end donation?
BCL-2/IgH
| BCL-1/IgH
71
How common is integration of ESCs?
1/10,000 to 1/100,000 recombination events
72
Where do ESCs reintegrate?
Cryptic RSSs upstream of an oncogene
73
How do chromosome translocations cause cancer?
Bring oncogenes under control of strong promoter
74
What unpublished data is there for ESCs causing cancer?
Cut and run of ESC with RSS bound to RAGs producing ssDNA cuts to produce free ends
75
How has the link between chromosome translocation and Class switch recombination been proved?
AID KO mice can produce no Myc-IgH translocation even when stimulated with pristine and IL6 translocation stimulants
76
What does AID overexpression cause?
Cancer
77
How are chromosome translocations protected against?
Ku70:Ku80 complex hold cut ends for NHEJC to seal
