1. IMMUN 441 Week 4 AC Quiz Section

3. DNA Transcription and Translation
Nucleus 5’
Germline DNA 3’
Pre-mRNA
Polyadenylation
Exon 1
Intron 1
Exon 2
Intron 2
Exon 3
AAAAAA
Splicing
AAAAAA
Cytosol
mRNA
Open Reading Frame
AAAAAA
Protein

4. DNA Gene Rearrangement5’
Germline DNA
Gene A
Gene B
Gene C
Gene D 3’
Permanently Lost
Germline DNA
Gene A
Gene D

5. V(D)J Recombination

6. Structure of the BCR loci
What are the proteins that carry out VDJ recombination?
Which cell types are they expressed in?
What would happen if a mutation caused RAG to have off target effects?

7. Step 1

8. Step 1
Step 2

9. Step 1
Step 2
Step 3

10. Step 1
Step 2
Step 3
Step 4

11. Step 1 Step 2 Step 3 Step 4 Step 5
Constant region of the heavy or the light chain determines the class?
Step 5

12. -α chain variability encoded in recombined V-J segments
What’s the difference between VDJ recombination in B cells versus T cells
TCR Recombination:
-α chain variability encoded in recombined V-J segments
-β chain variability encoded in recombined D-J segments
-TCR rearrangement utilizes the same proteins and mechanisms involved in Ig rearrangement

13. 12/23 rule Why is it important to have RSS?
Why can’t 23 and 23 come together or vice-versa?
What would happen if you had RSS elsewhere in the genome?

16. Ku: DNA repair protein required for Ig and TCR gene rearrangement
-forms a ring around the DNA and recruits DNA-PK

17. Junctional Diversity DNA-PK: recruits and activates Artemis
Artemis: nuclease that nicks hairpins at a random site

18. Junctional Diversity
TdT: inserts nontemplated nucleotides (N-nucleotides) between gene segments in V-regions

19. Junctional Diversity
DNA ligase: enzyme that joins ends of dsDNA

20. N-nucleotide addition during imprecise coding joint resolution can yield junctional diversity
Artemis cleaves hairpin, yielding P nucleotides V D
TdT adds N nucleotides V D
DNA ligase and repair joins gene segments V D
Junctional Diversity
This is only productive if new nucleotides are added in sets of 3 (D reading frame is preserved)

21. Productive vs. non-productive N-nucleotide addition during imprecise coding joint resolution: Frameshift mutations
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA AGT AAC TAT GAT GAA AGG
GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG
A N N E L I S E S N Y D E R V D

22. A N N E L I S E S N Y D E R Scenario 1:
Addition of 1 N-nucleotide by TdT
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA C AGT AAC TAT GAT GAA AGG
GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG
A N N E L I S E S N Y D E R V D

23. A N N E L I S E Q STOP Scenario 1:
Addition of 1 N-nucleotide shifts reading frame of remaining D gene, resulting in coding for truncated protein
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CAG TAA CTA TGA TGA AAG
GCU AAU AAC GAG CUA AUU AGC GAA CAG UAA CUA UGA UGA AAG
A N N E L I S E Q STOP V D

24. A N N E L I S E S N Y D E R Scenario 2:
Addition of 2 N-nucleotides by TdT
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CG AGT AAC TAT GAT GAA AGG
GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG
A N N E L I S E S N Y D E R V D

25. A N N E L I S E R V T M M K Scenario 2:
Addition of 2 N-nucleotides shifts reading frame of remaining D gene, resulting in coding for non-functional protein
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CGA GTA ACT ATG ATG AAA
GCU AAU AAC GAG CUA AUU AGC GAA CGA GUA ACU AUG AUG AAG
A N N E L I S E R V T M M K V D

26. A N N E L I S E R V T M M K Scenario 2:
Addition of 2 N-nucleotides shifts reading frame of remaining D gene, resulting in coding for non-functional protein
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CGA GTA ACT ATG ATG AAA
GCU AAU AAC GAG CUA AUU AGC GAA CGA GUA ACU AUG AUG AAG
A N N E L I S E R V T M M K
Nonsense amino acids yield a non-functional protein domain V D

27. A N N E L I S E S N Y D E R Scenario 3:
Addition of 3 N-nucleotides by TdT
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CGA AGT AAC TAT GAT GAA AGG
GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG
A N N E L I S E S N Y D E R V D

28. A N N E L I S E R S N Y D E R Scenario 3:
Addition of 3 N-nucleotides maintains original reading frame of remaining D gene, resulting in coding for nearly identical protein
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CGA AGT AAC TAT GAT GAA AGG
GCU AAU AAC GAG CUA AUU AGC GAA CGA AGU AAC UAU GAU GAA AGG
A N N E L I S E R S N Y D E R V D

29. A N N E L I S E R S N Y D E R Scenario 3:
Addition of 3 N-nucleotides maintains original reading frame of remaining D gene, resulting in coding for nearly identical protein
Region
Amino
Acid
mRNA
DNA
(sense)
GCT AAT AAC GAG CTA ATT AGC GAA CGA AGT AAC TAT GAT GAA AGG
GCU AAU AAC GAG CUA AUU AGC GAA CGA AGU AAC UAU GAU GAA AGG
A N N E L I S E R S N Y D E R
New sequence diversity!
(Non-germline-encoded)
=Junctional diversity V D

30. Unique features of the BCR

31. Ig somatic hypermutation (SHM) and Isotype class-switching (CSR) occurs in response to B cell activation by its cognate antigen
IgD
IgM
Sees antigen
SHM
Mature Naïve B cell
Mature Activated B cell
CSR
Would these mechanisms be considered anticipatory?
IgG
Secretion of soluble Ig
Class-switched B cell

32. Alternative splicing of BCR in mature naïve B cells allows coexpression of IgM and IgD isotypes
Is this permanent class switching, why or why not?

33. Alternative splicing of BCR in activated B cells allows coexpression of transmembrane and secreted Ig

34. Somatic Hypermutation and Class Switch Recombination both rely on AID

35. Multiple potential outcomes following AID mutation
Copying of the uracil
Templated replication
U recognized as T
Opposite strand = A
Transition

36. Multiple potential outcomes following AID mutation
Copying of the uracil
Removal of the uracil
Templated replication
U recognized as T
Opposite strand = A
Any base possible
Nontemplated replication
Does somatic hypermutation always result in higher affinity receptors? What happens to receptors with deleteriously mutations?
Transition
Transition or Transversion

37. Class switching
Why does the class matter?
What’s an example?

38. Class switching is initiated by AID targeting of switch regions

39. Class switching is initiated by AID targeting of switch regions

40. Class switching results in permanent isotype constant region excision
Intronic rearrangement = always productive

41. Class switching results in permanent isotype constant region excision

42. (P- and N- nucleotides) (⅓ productive) Somatic hypermutation (SHM)
Event
Processes
Pre- or Post-Ag?
B or T cells?
Nature of Change
Key Enzymes
DNA recombination
Pre-Ag
Both
Irreversible
Junctional diversity
(P- and N- nucleotides)
(⅓ productive)
Somatic hypermutation (SHM)
Point mutations
Post-Ag
B cells
AID, UNG
Class-switch recombination (CSR)
(all productive)
AID, UNG, APE-1
IgM/IgD expression
Differential
RNA splicing
Reversible (regulated)
Spliceosome
Membrane bound/secreted form
RAG1/2, TdT, Ku70:Ku80, DNA-PK, Artemis, DNA ligase
V(D)J recombination