Recombination & B cell Development Flashcards
How does recombination work?
DNA rearranges in developing B cells to bring one of many Vs together with the correct D so that the unit could be copied into messenger RNA
Ex: one V, one D and one J make up the VH domain gene
How do you make heavy chains using recombination?
- B cell first brings one random D segments close to one J (DNA)
- The DNA is cut and intervening DNA is discarded and the ends are joined
- It then brings a V segment up to the recombined DJ and repeats the cutting and joining process. (DNA)
- Then entire region from VDJ until through end of the delta constant region gene can be transcribed into RNA.
- This primary RNA transcript can be processed using alternative polyA sites and splicing - first to make VDJ-mu and later to make VDJ-mu and VDJ-delta
How do you make light chains using recombination?
Gene rearrangement is like heavy chains, except there are only V and J segments, no D (and only one C domain gene)
How does somatic variation contribute to diversity?
The cell has randomizing mechanism for production of V-D and D-J joints. The production of these joints is sloppy.
- First, EXONUCLEASES can chew away a few nucleotides after the DNA is cut but before the two gene segments (VD or DJ) are joined.
- Second, the cell can add a few nucleotides as well with an enzyme called TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) which doesn’t use a template so its addition are random.
How does somatic hypermutation work?
Recombined VDJ unit is ‘hypermutable’: each time a B cell divides after antigenic stimulation there is a good chance one of the daughters will make a slightly different antibody
What is the job of RAG1 and RAG2? What process do they work in?
RECOMBINATION
They are the enzymes that do the recombination of antibody and T cell receptor DNA (recombinases). they first bind splice signals to the right of a D segment and to the left of a J segment, pull them together and then cut and splice. Then they do it again with V.
What happens if RAG is knocked out?
No B or T cells can be produced = Omenn Syndrome
What does TdT do? What process does it work in?
SOMATIC VARIATION
It is an enzyme that adds a few nucleotides to a D-J or V-DJ joint. Terminal deoxynuclotidyl transferase (TdT) doesn’t use a template so its additions are random.
What is the job of AID? What process does it act in?
SOMATIC HYPERMUTATION
- Activation-Induced (Cytidine) Deaminase (AID) converts random cytosines in the CDR gene regions to uracil.
- So a C:G pair becomes a uracil: guanine mismatch
- The uracil bases are excised by the repair enzyme uracil-DNA glycosylase
- Error prone polymerases then fill in the gap, creating mostly single-base mutation so at the end of cell division one daughter may be making a different antibody. This is good because it will end up making Ab diversity and we can select for a better match
What is Allotypic Exclusion? What is its role in the generation of a functional B cell receptor (BCR)?
- Sloppy recombination can result in frame-shift mutation. Sometimes heavy and light chain genes are abortively rearranged in B cells. When this happens, the B cell tries again with another allele. So although any one cell is theoretically capable of making 2 heavy chains and 4 light chains, it only makes one of each.
- A B cell will try to rearrange each allele just once, but when a rearrangement is detected as faulty (stop codon, etc.), or when an anti-self receptor has been displayed, if the RAG genes are still active it can try again. Sometimes this results in a successful cell. This is called receptor editing/productive rearrangements.
How does RNA splicing and polyA site selection can dictate what isotype is produced and whether it is membrane bound?
If M1 M2 is present at the end of a chain it will be membrane bound. If it is cut at mu it will be IgM. If it is cut at delta it will be IgD, etc.
Describe the process of antibody class switching:
- After activation, B cell switch from membrane-bound IgM and IgD to secreted IgM by differential splicing.
- As the activated B cells continue to divide, they class switch to production of IgG by DNA rearrangement (again).
- Activated B cells may continue to class switch to produce IgE or IgA by further DNA rearrangement.
- In all cases the L chain and the VH domain stays the same but the C (constant) region of the H (heavy) chain changes
What is the order of antibody production?
M & D (membrane bound) –> secreted M –> IgG –> IgE and IgA [Mom & Dad Get it Every Afternoon]
What is the process of B cell Maturation that occurs in the bone marrow?
- Stem cell (not recombined)
- Early pro-B cell (not recombined)
- Late pro-B cell (DJ H region recombined)
- Large pre-B cell (VDJ H region recombined)
- Small pre-B cell (VDJ H region recombined)
- Immature B cell (VDJ H region recombined, VJ L region recombined)
What is the process of B cell maturation that occurs in the secondary lymphoid organs and circulation?
- Immature B cell (IgM+, IgD-) - Leaves bone marrow and enters peripheral circulation
- Immature B cell (IgM high, IgD low) - Alternative splicing to give both delta and mu chains. Gains access to primary lymphoid follicle and matures.
- Mature naive B cell (IgM low, IgD high) - Enters circulation and binds specific antigen in lymphoid tissue draining infection.
- Antigen-activated B lymphoblast - IgM only - Alternative splicing to secrete Ig Isotype switching, Somatic hypermutation
- Antibody secreting plasma cell (Secretes IgG and IgM) - Fighting the current infection
- Memory Cell (IgG) - Preparing for future infection
