Junctional Diversity, N-Nucleotide Addition and Somatic Hypermutation – Lymphocyte Development

00:01 So let’s look at this process of recombinatorial inaccuracy.

00:07 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.

00:18 CCC, CCC in the D segment and then a codon TGG in the J segment.

00:29 When the recombination occurs, it may be that the splice junction is taking the codon CCC and putting it next to TGG.

00:42 Now CCC is the codon for the amino acid Proline, and TGG is the codon for the amino acid Tryptophan.

00:51 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.

01:02 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.

01:12 So in this example, the B-cells use that CCC codon, so still be a Proline.

01:19 But because the splice point is slightly different, instead of the next codon being TGG, it’s CGG.

01:28 That means that instead of having a Tryptophan, there is an Arginine amino acid at that particular position.

01:34 And in the third example shown here, you can see that the second codon is CCG which again encodes the Proline.

01:42 So we have CCC and CCG both encoding Proline amino acids.

01:48 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.

02:00 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.

02:32 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.

02:43 So there’s an additional G, A and T.

02:46 Three additional nucleotides, non-templated nucleotides.

02:50 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.

03:00 And our enzymes are quite good because they actually usually do what they say they’re going to do.

03:04 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.

03:16 What you can see here is the third type of variability that allows antibodies to be incredibly diverse - somatic hypermutation.

03:28 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.

03:45 Clones of B-cells were isolated and the DNA sequenced.

03:50 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.

04:06 In Clone 2, there were no somatic hypermutations.

04:11 Whilst in Clone 3, there was a mutation immediately adjacent to CDR3 of the light chain Variable region.

04:20 At the far right of the slide, you can see the affinity of these antibodies.

04:26 These antibodies are of relatively low affinity.

04:30 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.

04:53 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.

05:08 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.

05:17 And with a third immunization with the same antigen, even more mutations accumulating.

05:23 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.