So let’s look at this process
of recombinatorial inaccuracy.
So, as you can see on the left, we
have the germline DNA, just showing
you the D segment and the J segment,
and showing you three codons.
CCC, CCC in the D segment and
then a codon TGG in the J segment.
When the recombination occurs, it may be that the splice
junction is taking the codon CCC and putting it next to TGG.
Now CCC is the codon for the amino acid Proline,
and TGG is the codon for the amino acid Tryptophan.
So in this particular recombination,
you’ll have a Proline and a Tryptophan
next to each other in the amino acid
sequence, but it doesn’t have to be.
For example in another B-cell using
exactlt the same D segment and exactly
the same J segment, the splice
junction may be slightly different.
So in this example, the B-cells use
that CCC codon, so still be a Proline.
But because the splice point is slightly different,
instead of the next codon being TGG, it’s CGG.
That means that instead of having a Tryptophan, there
is an Arginine amino acid at that particular position.
And in the third example shown here, you can see that
the second codon is CCG which again encodes the Proline.
So we have CCC and CCG both
encoding Proline amino acids.
So you can see that by this very basic kind of
technique in a way, of just slightly changing
where the splice junction is, it can create
additional diversity for the antibody molecule.
What about N-nucleotide addition?
Well, when the DNA is cut during D to J
recombination, and V to D recombination
in the heavy chain, and J… V to J
recombination in the light chain; before the
DNA is re-spliced, the enzyme Terminal
deoxynucleotidyl Transferase can pick up
one or more nucleotides and insert them
at the place where the DNA has been cut.
So in this example, TdT is picking up a G, it’s picking up a T,
and picking up an A, and putting those in the splice junction.
So there’s an additional G, A and T.
Three additional nucleotides,
They’re called non-templated because that… those
nucleotides are not determined by the DNA sequence,
they’re just being picked up randomly by this
enzyme, Terminal deoxynucleotidyl Transferase.
And our enzymes are quite good because they actually
usually do what they say they’re going to do.
Terminal - at the ends, deoxynucleotidyl
- it’s picking up a deoxynucleotide,
in this example a G, an A or T, and transferring
it onto the cut ends of the DNA.
What you can see here is the third
type of variability that allows
antibodies to be incredibly diverse - somatic hypermutation.
In front of you, you have the results of an
experimental approach to looking at the accumulation
of point mutations in the DNA of the immunoglobulin
heavy and light chain Variable region genes.
Clones of B-cells were
isolated and the DNA sequenced.
If you look at Clone 1, you can see that
in the CDR3 of the heavy chain, there
is a point mutation that has occurred,
indicated by the vertical black bar.
In Clone 2, there were no
Whilst in Clone 3, there was a mutation immediately
adjacent to CDR3 of the light chain Variable region.
At the far right of the slide, you can
see the affinity of these antibodies.
These antibodies are of
relatively low affinity.
But as the primary immune response develops,
looking now at Day 14; isolating different
clones at Day 14 and sequencing them, you
can see that there are more vertical
bars have appeared, in other words there are
more point mutations that have accumulated,
both in the heavy chain Variable region
and the light chain Variable region.
And you can see, a couple mutations in
CDR2 of the heavy chain Variable region
and a number of mutations within CDR1
or adjacent to CDR1 in the light chain.
If you re-immunize the mouse a little
bit later with the same antigen and
develop a secondary immune response, you
can see more mutations accumulating.
And with a third immunization with the same
antigen, even more mutations accumulating.
And what is important, is if you look at the right hand
side, that column where the affinity is shown, you can see
that the result of this somatic hypermutation is to increase
the affinity, the strength of binding of these antibodies.
So somatic hypermutation is very important
in creating additional diversity, but also
in helping to improve or increase the strength
of binding, the affinity of the antibody.