MCB 408 Exam 2 Questions with Correct Answers Graded A+
Five mechanisms generate antibody diversity in naive B cells - Answers - multiple gene segments V(D)J:
assembling different gene segments into one generates diversity. (1987 Nobel Prize Susumu Tonegawa
- P (palindromic) nucleotide addition: templated nucleotide addition between joints resulting from
asymmetrical cleaving of hairpin structures.
- Exonuclease trimming: sometimes occurs at junctions, losing nucleotides and changing reading frames
(trim pieces back)
- Nontemplated N nucleotide addition: mediated by TdT activity, adding in random nucleotides between
joints
- Heavy chain/light chain combinatorial diversity. combine in different ways
Recall that Ig proteins consist of: - Answers - 2 identical heavy chains
- 2 identical light chains, which can be either kappa or lambda
Each set of gene families are encoded on separate chromosomes - Answers - kappa genes on human
chromosome 2, mouse chromosome 6
- lambda genes on human chromosome 22, mouse chromosome 16
- heavy chain genes on human chromosome 14, mouse chromosome 12
Variable (V), Diversity (D), joining (J), and constant (C) region gene segments recombine to form a
complete Ig gene - Answers * no D segments in light chains -> it is only in heavy chain
- VJ recombination: kappa light chain
- heavy chain recombination occurs first
- Lambda light-chain genes pair each J segment with a particular C segment
- VDJ recombination
- V segment (amino acids 1-101) -> CDRs 1 and 2
- D segment (amino acids 102-106 approx.) and J segment (amino acids 107-123 approx.) -> CDR3
- Regions outside of CDRs are called "Framework regions"
~120 residues
BCR V(D)J recombination occurs in the bone marrow - Answers - Early Pro-B: DJ H chain recombination
and start od V-DJ H chain recombination
,- Late Pro-B: V-DJ H recombination
- Pre-B: u H chain expressed as pre-BCR. several rounds of cell division
- VJ L chain recombination
- Immature B: mIgM expression. Negative selection -> deletion and receptor editing
V(D)J recombination is directed by the Recombination signal sequence (RSS) - Answers Recombination
signal sequences flank each antibody gene segment
- Each has a conserved nonamer (9 bases) and heptamer (7 bases) sequence
- in between the nonamer/heptamer lies either a 12 or 23 bp spacer sequence
- the spacing and arrangement dictates that a 12 bp RSS must pair with 23 bp RSS for recombination to
occur (the "12/23 Rule"). the identity of bases is spacer is not conserved, length is conserved
- this prevents two different genes coding for the same region from recombining (ex. V-V
recombination). its always V,D,J
Recombination Signal Sequences (RSS) flank each antibody gene segment - Answers Germline heavy
chain DNA - the DJ rearrangement occurs first
Then the V-D rearrangement occurs
V(D)J segments are joined by the RAG1/2 recombinase - Answers RAG1/2 recombinase is essential for
this process
- only expressed in lymphocytes (T cells and B cells)
- not something you want around your other cells
- very tightly regulated!
Regulation of V(D) J gene recombination involves chromatin alteration - Answers - only in B cells and T
cells
- the catalytic activity of RAG1/2 occurs in an extraordinarily complex nuclear environment
- RAG1/2 binding is affected by epigenetic modifications on histones associated with target sequences.
recognizes cell cycle states.
- has to be very well regulated
- histone acetylation or methylation affects accessibility to enzymatic activity in chromatin
- because the V, D, J gene segments are so spread out along the chromosome, higher order chromatin
structure must play a role.
,Steps of V(D)J segments being joined by the RAG1/2 recombinase - Answers 1. lymphoid specific (B cells
and T cells)
2. Functions as a tetramer
3. introduces a nick at the 5' end of the RSS heptamers adjacent to the coding regions
4. removes the loop of intervening DNA
5. hairpin loop formation on strand -> then linked
6. subsequent steps include double-stranded break and repaired by other enzymes.
V: 23-base pair spacer
J: 12-base pair spacer
RAG1 (larger): nonamer binding domain and catalytic DNA cleavage
- E3 ubiquitin ligase
- H3 interaction (modify and bind H3)
- heptamer binding (forms a complex)
- RAG2 interaction (primary cleavage activity)
RAG2 (smaller)
- DNA cleavage
- DNA binding
- Binds H3K4me3
- Enhances catalytic activity (cleaves)
- makes sure everything is bound then it can cleave
- then recognizes features of cell cycle regulation
The addition of nucleotides at coding junctions increases diversity (junctional diversity) - Answers
Addition of Palindromic, "P" nucleotides. fills in the sequences
, - Terminal deoxynucleotidyl transferase (TdT), adds further Non-templated 'N' nucleotides. increases
diversity of heavy chain CDR3.
- CDR3 is the most variable!
- unproductive items are common
RAG1/2 (specific to lymphocytes) - Answers Function in V(D)J recombination: antigen receptor gene
recombinase complex. DNA cleavage is mediated by RAG1. Epigenetic targeting is directed by RAG2
Immunological consequences of protein deficiency: Sever combined immuno-deficiency (SCID)
Terminal deoxyribonucleotidyl transferase (TdT) (specific to lymphocytes) - Answers Function in V(D)J
recombination: adds nontemplated (N) nucleotides to V-D and D-J joints of Ig heavy chaim and all joints
of TCR chains in a template-independent manner
Immunological consequences of protein deficiency: reduced N-nucleotide addition is seen at coding
joints
V(D)J recombination occurs in a series of well-defined steps - Answers - RAG proteins bind to RSSs and
cleave the DNA
- other proteins process the hairpin loops that form after RAG processing
- leftover signal joint is later degraded
- products include a recombined coding joint which may have P and N nucleotides
- ultimately, V(D)J will encode the protein sequence of the variable domains, including the CDR loops in
a BCR or antibody
Heavy chain recombination is followed by a test... if successful light chain recombination will start -
Answers Early Pro-B and Late Pro-B is where heavy chain recombination occurs
Pre-B: starts light chain recombination
Following successful* heavy chain recombination, the heavy chain becomes a "PreB cell receptor" -
Answers - heavy chain is assembled into a surrogate light composed of VpreB and lambda5.
- this ends heavy chain recombination and signals
1. proliferation
2. the start of light chain recombination
Five mechanisms generate antibody diversity in naive B cells - Answers - multiple gene segments V(D)J:
assembling different gene segments into one generates diversity. (1987 Nobel Prize Susumu Tonegawa
- P (palindromic) nucleotide addition: templated nucleotide addition between joints resulting from
asymmetrical cleaving of hairpin structures.
- Exonuclease trimming: sometimes occurs at junctions, losing nucleotides and changing reading frames
(trim pieces back)
- Nontemplated N nucleotide addition: mediated by TdT activity, adding in random nucleotides between
joints
- Heavy chain/light chain combinatorial diversity. combine in different ways
Recall that Ig proteins consist of: - Answers - 2 identical heavy chains
- 2 identical light chains, which can be either kappa or lambda
Each set of gene families are encoded on separate chromosomes - Answers - kappa genes on human
chromosome 2, mouse chromosome 6
- lambda genes on human chromosome 22, mouse chromosome 16
- heavy chain genes on human chromosome 14, mouse chromosome 12
Variable (V), Diversity (D), joining (J), and constant (C) region gene segments recombine to form a
complete Ig gene - Answers * no D segments in light chains -> it is only in heavy chain
- VJ recombination: kappa light chain
- heavy chain recombination occurs first
- Lambda light-chain genes pair each J segment with a particular C segment
- VDJ recombination
- V segment (amino acids 1-101) -> CDRs 1 and 2
- D segment (amino acids 102-106 approx.) and J segment (amino acids 107-123 approx.) -> CDR3
- Regions outside of CDRs are called "Framework regions"
~120 residues
BCR V(D)J recombination occurs in the bone marrow - Answers - Early Pro-B: DJ H chain recombination
and start od V-DJ H chain recombination
,- Late Pro-B: V-DJ H recombination
- Pre-B: u H chain expressed as pre-BCR. several rounds of cell division
- VJ L chain recombination
- Immature B: mIgM expression. Negative selection -> deletion and receptor editing
V(D)J recombination is directed by the Recombination signal sequence (RSS) - Answers Recombination
signal sequences flank each antibody gene segment
- Each has a conserved nonamer (9 bases) and heptamer (7 bases) sequence
- in between the nonamer/heptamer lies either a 12 or 23 bp spacer sequence
- the spacing and arrangement dictates that a 12 bp RSS must pair with 23 bp RSS for recombination to
occur (the "12/23 Rule"). the identity of bases is spacer is not conserved, length is conserved
- this prevents two different genes coding for the same region from recombining (ex. V-V
recombination). its always V,D,J
Recombination Signal Sequences (RSS) flank each antibody gene segment - Answers Germline heavy
chain DNA - the DJ rearrangement occurs first
Then the V-D rearrangement occurs
V(D)J segments are joined by the RAG1/2 recombinase - Answers RAG1/2 recombinase is essential for
this process
- only expressed in lymphocytes (T cells and B cells)
- not something you want around your other cells
- very tightly regulated!
Regulation of V(D) J gene recombination involves chromatin alteration - Answers - only in B cells and T
cells
- the catalytic activity of RAG1/2 occurs in an extraordinarily complex nuclear environment
- RAG1/2 binding is affected by epigenetic modifications on histones associated with target sequences.
recognizes cell cycle states.
- has to be very well regulated
- histone acetylation or methylation affects accessibility to enzymatic activity in chromatin
- because the V, D, J gene segments are so spread out along the chromosome, higher order chromatin
structure must play a role.
,Steps of V(D)J segments being joined by the RAG1/2 recombinase - Answers 1. lymphoid specific (B cells
and T cells)
2. Functions as a tetramer
3. introduces a nick at the 5' end of the RSS heptamers adjacent to the coding regions
4. removes the loop of intervening DNA
5. hairpin loop formation on strand -> then linked
6. subsequent steps include double-stranded break and repaired by other enzymes.
V: 23-base pair spacer
J: 12-base pair spacer
RAG1 (larger): nonamer binding domain and catalytic DNA cleavage
- E3 ubiquitin ligase
- H3 interaction (modify and bind H3)
- heptamer binding (forms a complex)
- RAG2 interaction (primary cleavage activity)
RAG2 (smaller)
- DNA cleavage
- DNA binding
- Binds H3K4me3
- Enhances catalytic activity (cleaves)
- makes sure everything is bound then it can cleave
- then recognizes features of cell cycle regulation
The addition of nucleotides at coding junctions increases diversity (junctional diversity) - Answers
Addition of Palindromic, "P" nucleotides. fills in the sequences
, - Terminal deoxynucleotidyl transferase (TdT), adds further Non-templated 'N' nucleotides. increases
diversity of heavy chain CDR3.
- CDR3 is the most variable!
- unproductive items are common
RAG1/2 (specific to lymphocytes) - Answers Function in V(D)J recombination: antigen receptor gene
recombinase complex. DNA cleavage is mediated by RAG1. Epigenetic targeting is directed by RAG2
Immunological consequences of protein deficiency: Sever combined immuno-deficiency (SCID)
Terminal deoxyribonucleotidyl transferase (TdT) (specific to lymphocytes) - Answers Function in V(D)J
recombination: adds nontemplated (N) nucleotides to V-D and D-J joints of Ig heavy chaim and all joints
of TCR chains in a template-independent manner
Immunological consequences of protein deficiency: reduced N-nucleotide addition is seen at coding
joints
V(D)J recombination occurs in a series of well-defined steps - Answers - RAG proteins bind to RSSs and
cleave the DNA
- other proteins process the hairpin loops that form after RAG processing
- leftover signal joint is later degraded
- products include a recombined coding joint which may have P and N nucleotides
- ultimately, V(D)J will encode the protein sequence of the variable domains, including the CDR loops in
a BCR or antibody
Heavy chain recombination is followed by a test... if successful light chain recombination will start -
Answers Early Pro-B and Late Pro-B is where heavy chain recombination occurs
Pre-B: starts light chain recombination
Following successful* heavy chain recombination, the heavy chain becomes a "PreB cell receptor" -
Answers - heavy chain is assembled into a surrogate light composed of VpreB and lambda5.
- this ends heavy chain recombination and signals
1. proliferation
2. the start of light chain recombination
