Let’s take a few moments to look in a little bit more detail at
how this genetic recombination process actually is mediated.
Really depends on two things,
or requires two things.
Requires a number of different enzymes that catalyze the
recombination process, but it also requires recognition
sequences for these enzymes, in other words substrates
for these enzymes that are within the DNA sequence.
They’re not in the part of the DNA that encodes the
protein but they lie immediately adjacent to that part.
And they’re called Recombination
Signal Sequences or RSS.
And the enzymes that recognize these
Recombination Signal Sequences,
their nucleotide sequences as I say,
flanking the V, D and J segments;
the enzymes that recognize, or the
two most important enzymes that
recognize these Recombination Signal
Sequences are called RAG-1 and RAG-2.
So heavy chain VDJ recombination, and likewise light chain
VJ recombination is mediated by the recombinase enzymes RAG-1
and RAG-2; stands for Recombination Activating Genes, even
though they’re actually proteins that come from those genes.
And they recognize these Recombination Signal Sequences or
RSS, which are nucleotides flanking the V, D and J gene segments.
And as you can see from this particular
picture, the V segment has an RSS
immediately three prime or downstream
of the protein coding sequence.
The D segment has two
RSSs, one either side.
And the J segment has a signal RSS five prime
or immediately upstream of its sequence.
So following successful heavy chain
recombination of VDJ, the light chain is
then recombined, because an antibody needs
both a heavy chain and a light chain.
And here we can see the Variable region
of the kappa (k) light chain, around about
40 different Variable segments for the light
chain in the k… for the k light chain.
And five or so Joining gene segments.
And this particular B-cell has decided to recombine its k
light chain using a Vk2 and recombining it next to Jk2.
And then just as we saw for the heavy chain,
it will make a primary RNA transcript,
it will process that into message and
then translate that into protein.
And again the CDR3 in the light chain is the
most variable because it’s a combination
of any one out of those 40 Vs, put next to
one of those five different J gene segments.
So the recombination occurs in both the
heavy chain and the light chain, and that
recombination being a fairly random process
is not actually always successful.
Sometimes it doesn’t work.
So there might be a out of reading frame sequence
generated or maybe a stop codon is generated.
So in some ways, it’s a bit of a wasteful
process given that it doesn’t always work.
But it’s an incredibly powerful process because it means that
from a handful of genes, you can
make millions, and millions, and
millions and millions of different antibody molecules; and
likewise many, many million different T-cell receptor molecules.
So the sequence of events is that there are
two attempts at recombining the heavy chain.
Remember we have two
copies of each chromosome.
So you’ve inherited one copy of your immunoglobulin heavy
chain genes from your mom and another copy from your dad.
And given that this random process is not always
successful, maybe you fail to make a B-cell,
fails to make a productive, what we call
productive recombination on one of the chromosomes.
All’s not lost, it can
try the other chromosome.
As long as one attempt is successful, then it will go on,
the B-cell will go on and recombine the light chain genes.
It starts off with the k light chain.
Again there are two attempts,
because you have two versions.
One you’ve inherited from
your mom, one from your dad.
In fact, for the light chains, there’s a total
of four possible attempts, because as well as
having the k light chain genes as we’ve already
heard, we have the lambda (λ) light chain genes.
So if both recombinations on the k
chain are unsuccessful, there’s still
hope left, there’s still a chance,
because there are two λ chain gene loci.
So hopefully the B-cell will be successful
in both the heavy chain recombination,
one out of two choices, light chain
recombination, one out of four choices.
And produce a functional antibody molecule
that will then be put on the cell surface.
It's important that B-cells are
specific for one single antigen.
So for example, you’ll have one B-cell
that’s specific for Staphylococcus, another
for Streptococcus, another for Candida,
another for Cytomegalovirus and so on.
And in order to ensure that the B-cell
is only specific for a single antigen,
you need to just have one heavy chain
sequence and one light chain sequence.
And that is ensured by a process that
is referred to as allelic exclusion.
The result of allelic exclusion is that
if the first attempt at recombining
a heavy chain is successful, recombination
on the other chain is prevented.
It’s not allowed.
So as the first recombination is
successful, you make a protein, there’s
feedback mechanism that prevents recombination
on the other heavy chain locus.
Likewise, for the light chain recombination, if there’s
a successful recombination of the light chain, either
the k light chain or the λ light chain, it prevents
subsequent recombination of other light chain loci.
So, allelic exclusion ensures that there
isn’t a mixture of different heavy chains
or different light chains for a given B-cell;
just one heavy chain, one light chain.
There’ll be millions of copies,
but they’ll all be identical.