Generation of Antibody Diversity. The big question:how do you get all those different antibodies? Two other big questions- getting different classes with the same variable regions, and getting secreted and membrane-bound forms of the same class
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85X 4 possible combinations=340
134X 13X 4 possible combinations =6968
Total = 343X6968= 2,390,024- a lot, but not gazillions.
Southern blot, using J region as a probe. In a germ line culture, the probe only finds the J region. In a B cell line, it finds the rearranged V region as well. Further cloning and sequencing verified this result.
Fig. 22-9 for more information
Joining flexibility adds more diversity, and also contributes to nonproductive rearrangements
These are the ends of the J and V regions after any P-nucleotide addition has taken place, and also N-nucleotide addition (in H chain)
Allelic exclusion: successful rearrangement of 1 allele excludes rearrangement of the other allele.
The cell “knows” when it’s done an unproductive rearrangement!
Periodically remove cells and make monoclonals- after primary immunization, 2o, 3o immunizations
Sequence H and V chain mRNA from these monoclonals
Compare sequences and antibody affinity
Switch recombinase, signaled by cytokine
Consequences? Can’t go backwards!
These have been found
In eukaryotes, we make a primary transcript, that can then be cut- where it is cut is where we add a “polyA tail”
Mu really consists of six exons
Delta really conists of 5 exons