L9: Generation of Diversity Flashcards
What is the T cell receptor made up of?
2 chains, an alpha chain and a beta chain
What is the B cell receptor made up of?
A heavy chain and a light chain
There are 2 light chain genes: kappa and delta (one or the other is included in the BCR expressed by an individual B cell)
What processes generate diversity in both B cells and T cells?
- Multiple copies of each gene segment that makes up the variable joined in different combinations (combinatorial)
- Addition and subtraction of nucleotides by the recombination process (junctional diversity)
- Many different combinations of H and L chain V regions (or alpha and beta V regions) that pair to form the antigen binding site (combinatorial)
What additional processes add to BCR diversity?
- Somatic hypermutation occurring in the germinal center
How many antigen binding sites do TCRs and BCRs have? What are they made up of?
BCRs: 2 identical antigen binding sites; formed by the heavy chain and the light chain
TCRs: Singular antigen binding site; formed by the alpha and beta chains
Where are BCRs expressed?
On surface of B cell or secreted as antibody
Why is generation of diversity important?
Survival is dependent upon a wide range of antigen specific receptors, differing in their amino acids sequences at the antigen binding site
But encoding each receptor chain variant in the genome would require more genes than are present in the entire genome
What undergoes splicing in TCR and BCR genes?
Both the DNA itself as well as the primary RNA transcript
Describe Ig light chain gene construction
Light chain variable region (V_L) is constructed from 2 segments, a variable (V) and joining (J) gene segment; there is a leader peptide (L) that precedes the V gene segment
V and J gene segments in the genomic DNA are joined to form a complete V_L region exon → Light-chain C region (C_L) is encoded in a separate exon and is joined to the V_L exon by splicing of the light-chain RNA to remove the L-to-V and J-to-C introns
Describe Ig heavy chain gene construction
Heavy chain V region (V_H) is constructed from 3 gene segments: the diversity (D), V, and J segments
D and J gene segments join → V gene segment joins to the combined DJ sequence → forms a complete V_H exon → During RNA processing of heavy-chain RNA transcript, C-region exons, together w/ leader sequence, are spliced to the V_H domain sequence
How many gene segments are there for V, D, and J loci?
Multiple gene segments for each
For instance, for heavy chain there are 45 possible V regions
What is intragenic recombination mediated by?
Mediated by the RAG recombinases recognizing RSS (recombination signal sequence)
Describe what RAG recombinases do?
RAG recombinase components (RAG-1 and RAG-2) align the segments that will be spliced and excise the intervening DNA including the RSS sequence
Get a signal joint and a coding joint (this gives you VJ region; this is the spliced region that will be expressed)
Rejoining of DNA ends is done by DNA repair enzymes
Why does immunodeficiency occur if there is a problem w/ DNA repair enzymes?
Can’t complete DNA repair necessary for T and B cell diversity since can’t resolve the cuts that the RAG enzymes have made
Where are RAG-1 and RAG-2 expressed?
Only in developing lymphocytes
since don’t want it recombining genes in random
Is ligating at the ends of the signal joint and the coding joint precise?
At the signal joint, DNA ligase joins the blunt ends precisely
At the coding join, ligation occurs imprecisely, thus inducing additional variability y into the variable region
TdT
(Terminal deoxytransferase) Adds N (nontemplated) nucleotides at the coding joints; this is a random process
What can aberrant rearrangements lead to?
Chromosomal translocations and malignancy
The unique VDJ rearrangement aids in recognizing the presence of a lymphoid malignancy
In B cell development, does H chain or L chain gene rearrangement occur first?
VDH rearrangement of heavy chain occurs first
VJ rearragement of light chain then occurs
How do nonproductive rearrangements occur?
Added nucleotides at the junctions often disrupt the reading frame, causing a nonproductive rearrangement in 2/3
What happens when nonproductive rearrangements occur?
B cell progenitors that do not produce functional immunoglobulin never mature
If rearrangement in unsuccessful, what occurs?
Try again since there are 2 alleles
If get it wrong twice, there is cell death
Once able to express heavy chain protein, the rearrangement for light chain protein begins
What do all BCR constant regions start off as?
IgM
IgM and IgD seem to be expressed in early B cells
What is expressed on the surface of naive B cells?
IgM and IgD
These cell have not yet undergone class-switching
In germline TCR genes, are there D regions?
Alpha-chain locus has V and J
Beta-chain locus has V, D, and J
If you have defects in VDJ recombination in either RAG or in DNA repair enzymes, what cells are going to have defects?
Both B and T cells
Describe TCR rearrangement compared to BCR rearragement
Occurs by the same mechanisms
RAG-1/2 bind to RSS sequences and mediate DNA breakages
Rejoining is mediated by the same DNA repair enzymes
Addition of P and N nucleotides at joints occurs in the same way
Defects in VDJ recombination affect both T and B cells
Somatic hypermutation does not occur after TCR rearrangement
