Antibody Genes II Flashcards
Explain why we commonly write V(D)J instead of VDJ.
The light chain only comes from the joining of V and J, whereas the heavy chain comes from the joining of D and J followed by the joining of V with DJ (The variable domain region of heavy chain genes is composed of multiple V, multiple D and multiple J gene segments, the V region of light chains into V and J segements. The cell will choose one of its Vs, 1 D and 1 J to make the variable heavy chain domain gene region. )
Describe the somatic recombination model (“class switching”), which explains how antibodies of the same specificity (that is, with the same CDRs and idiotype) can be found in two or more different classes.
Class switching: single mature B cell starts by making IgM and IgD. Later, it may switch to IgG, IgE or IgA. In all cases, the V domain stays the same but the C region of the H chain changes. The cell with its H-chain VDJ combination together with its mu and delta, puts VDJ next to the C-region gene of gamma or epsilon or alpha and excises the intervening DNA. The new mRNA, the may be VDJ (alpha), VDJ(epsilon), VDJ(gamma). A cell which is making IgM can go on to make IgG. A cell making IgG CANNOT go back to making IgM because the mu gene is physically GONE. A cell can switch heavy chains, but cannot switch light chains.
Based on the (outdated) concept of “one gene-one H or L chain,” calculate the minimal number of genes required to code for a million different antibody molecules.
2000 genes (1000 L chains and 1000 H chains, 1000x1000= 1,000,000 antibody combining sites.
Show how breaking the variable region gene up into V, D and J subregions requires fewer genes
- V-D and D-J joins are “sloppy”. #1: Exonucleases chew away a few nucleotides after the DNA is cut but before the 2 gene segements (D to J, V to DJ) are joined. #2: terminal deoxynucleotidyl transferase (TdT) adds a few nucleotides without using a template, so its additions are random. Thus you CANNNOT predict the sequence at the joining area, called the “N region”. This produces a lot of diversity, but 2/3 times the N region will not be the correct length (incorrect # of bases added) and the frame-shift mutation occurs (nonsense codon that terminates transcription).
- A lot of diversity in the germ line (the individual V, D, J segements you are born with), a lot of diversity in the “slopping” variable V-J and V-D joining. Also, the recombined V(D)J unit is hypermutable: each time a B cell divides after antigenic stimulation, there is a good chance that one of the daughter cells will amek a slightly different antibody (selection of the best fitting mutants gradual increase of affinity during immune response).
Define somatic hypermutation
each time a B cell divides after antigenic stimulation, there is a good chance 1 of the daughter cells will make a slightly different antibody mutants that are better (or worse) affinities during immune response. This happens via activation-induced (cytidine) deaminase (AID) which converts a random Cytosine in CDR gene regions to uracil. C:G becomes U:G mismatch. Uracil removed by repair enzyme and error-prone DNA polymerases fill in the gap creating mostly single-base substitution mutations so at the end of cell division the daughter may be making a different antibody
distinguish hypermutation from the somatic mutation mechanism that produces N-region diversity.
N-region diversity: created by the “sloppiness” of V-D and D-J joining. 1: exonucleases chew away a few nucleotides after the DNA is cut but before the gene segments (D to J and V to DJ) are joined. 2: enzyme TdT adds nucleotides without a template (random!), you can’t predict the sequence at the joining area “N region”. Lots of diversity produced, but the price is frame shift mutations that occur frequently
Define somatic mutation
Alterations in DNA that occur after conception. Somatic mutations can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore are not passed on to children.
describe the essential difference between the somatic mutation and germ line hypotheses of immunological diversity.
There used to be two schools of thought about antibody diversity: one said that all the V genes were in the germ line; if you looked at a fertilized ovum you could predict all potential antibodies that potential individual could potentially make. The other said that only a few were there. It postulated that during embryonic lymphoid development these genes underwent repeated (somatic) mutation until a full complement of antibodies was produced. Both theories, it turns out, were right. As we’ve just discussed, a lot of our diversity is in the germ line (that is, in the individual V, D, and J segments you’re born with). Even more diversity is also generated by variable (“sloppy”) V/J and V/D joining.
