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Immunology Notes- BCR - Organization and Expression of
Lymphocyte Receptor Genes: BCR, taught by Agnieszka
Immunology (Montana State University)
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Lecture 7 Notes- Organization and Expression of Lymphocyte Receptor Genes: BCR
(Kuby Chapter 6)
Basic Structure of BCR:
- Y shaped protein
- 2 light chains on the outside (L) -> chains are identical
- 2 heavy chains on the inner portion (H) -> chains are identical
- Interchain disulfide bonds there connect the two heavy chains together as well as the
two heavy chains to the light chains
- 2 identical antigen bonding sites -> located at the top of the light and heavy chains
(noted by N terminal)
- Antibody effector function (notated by C terminal) -> located at the bottom of the two
heavy chains. Induced via the Fc region which interacts with complement proteins
Theories of the Immunoglobulin Gene Structure
- Germ-line theory -> There is only one gene for one protein.
- Somatic Hypermutation Theory -> A limited number of genes encode the V region, but
some unknown mutational mechanism is responsible for variation.
Multi-gene Organization of Ig Genes
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Variable (V) exon DNA
Constant (C) exon DNA
Gene segments that make up the variable exon -> arranged in clusters
- Variable (V) segments
- Diversity (D) segments
- Joining (J) segments
- Light chains are made up of V and J segments (k chains and � chain)
- Heavy chains are made up of V, D and J segments
Light Chains
- 2 types of light chains -> k and � chain
- V (Variable) exon -> V and J gene segments
� V region made up of only 3 segments (V�1-V�3)
- C (constant) exon ->
k V region -> one C exon
� V region -> four C exons (C�1-C�4)
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Heavy Chains
- V exon -> V, D and J segments
- C exon -> 8 Ch regions, one for each antibody isotope (class)
Cm, Cd, Cg (4 subclasses), Ce, Ca
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Each set of gene families are encoded on separate chromosomes
k genes on human chromosome 2, mouse chromosome 6
� genes on human chromosome 22, mouse chromosome 16
heavy chain genes on human chromosome 14, mouse chromosome 12
From Exons to Ig Proteins
- V(D)J Recombination -> Occurs at the DNA level, somatic recombination, lose
intervening DNA
- V exon joined to C exon -> Occurs at the RNA level, primary RNA transcript (RNA
splicing!!!!)
- Production of MIgD or MIgM -> Occurs at the RNA level, primary RNA transcript (alt
RNA splicing)
Gene Segment Multiplicity & Combinatorial Joining of Heavy and Light Chains
- The primary source of diversity! -> generates thousands of potential combinations
- Examples:
41 Vk x 5 Jk = 205 possible Vk exon sequences
33 Vlx 5 Jl = 165 possible Vlexon sequences
45 VH x 23 DH x 6 JH = 6210
H & L chain combinations: 5858 x (205 + 165) = 2.3 x 106
V(D)J Recombination
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This mechanism involves ->
RSS (recognition signal sequence)
RAG ½ (lymphocyte specific enzymes)
DNA PK, ligase, and oth. repairing enzymes
TdT (lymphocyte specific enzyme)
RSS -> nucleotide Sequence consists of heptamer (7) and nonamer (9) sequences separated by
a spacer (12bp = 1 DNA turn, 23bp = 2 DNA turns) sequence
- Flanks each gene segment!
Downstream of the V segment
Upstream of J segment
On each side of the D segment
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12/23 Rule -> 12 bp RSS MUST pair with 23 bp RSS. This ensures the order on gene
arrangement.
Four Basic Steps of V(D)J Recomb.
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1.
2.
3.
4.
Binding of RAG1/2 to RSS
Synapsis
Cleavage of signal & coding joints
Generation of functional variable region exon
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Note: V(D)J recombination can occur between segments transcribed in either the same or
opposite directions. Orientation of the actual gene segment is irrelevant.
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Steps 1-4 Visulaization
● Palindromic nucleotide addition
happens in both light and heavy
chains!
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RAG ½
- Recombination activation genes
- Are lymphocyte specific
- Necessary for V(D)J recombination
- RAG 1 and RAG 2
TdT -> Terminal
deoxynucleotidyl transferase
- Adds non-templated nucleotides (N nucleotides)
- Occurs in heavy chains only!
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Lymphocyte specific!
Junctional Diversity: Imprecise Joining of V, D, and J Gene Segments
- 3 mechanisms:
1. P nucleotide addition
- P = palindromic sequences added by DNA repair enzymes
2. Exonuclease trimming
- occurs in heavy chain
3. N nucleotide addition
- non-template encoded nucleotides added by TdT. Occurs only in
heavy chain
Non-productive Gene Rearrangements
- When VJ or VDJ creates stop codon or frameshift mutation -> results in non-fnx
protein = non-productive
Productive Arrangements
- In phase joining
5 Mechanisms to Create Antibody Diversity
- Multiple gene segments
- which gene segments are put together
- P nucleotide addition
- Exonuclease trimming
- N-nucleotide addition
- Combinatorial Diversity
- heavy/light chain pairing
* Junctional Diversity
BCR Expression
Allelic Exclusion
- Phenomenon whereby B cells only express 1 allele of the heavy chain & 1 allele of the
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light chain -> One allele is expressed while the other is silenced
Heavy and light chains of either mother or father can be expressed
Significance? Only 1 type of antibody can be expressed at 1 time
ROLE IN THE DEVELOPMENT OF B-LYMPHOCYTES
Proposed allelic exclusion mechanism:
- Non-productive rearrangements lead to cell death!
- 2 alleles for heavy chain = 2 tries for a productive rearrangement
- 4 alleles for light chain = 2 tries for k chain, 2 tries for l chains
PRE-BCR
- Heavy chain is present
- Surrogate light chain (SLC) is present -> made up of VpreB + �5
- The heavy chain are recombined and expressed first
- The heavy chain is paired up with the SLC
- Signaling through the pre-BCR results in the inhibition of heavy chain
recombination of the 2nd allele via transcriptional silencing of recombination
machinery, several rounds of proliferation, requires CD19
- Light chain recombination then begins -> the expression of the recombination
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machinery is re-initiated
Visualization on next page ->
Light Chain Receptor Editing
- Occurs in the light chains
- The B-cell uses the same allele more than once
- If the B-cell is autoreactive the cell can try 2nd arrangement
- Recombination machinery is turned back on
- Last ditch effort salvage the rearrangement
Codominance of mIgM & mIgD = Co-Expression
- Naive mature b-cells express both mem. Bound IgM and IgD
- This is due to alternative RNA splicing -> a way to produce a bunch of mRNA
from the low amount of human genes
- 2 ploy A signals -> Signals located near the 5’ end of the gene
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Expression of membrane vs. secreted Ig’s
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When Happen in
the Lifespan of a
B-cell?
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For Reference
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