Variable regions of the immunoglobulin gene

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The variable regions of the immunoglobulin gene can only be recombined to the joining regions, the variable regions in this case being V1,V2 and Vx they can only work downstream (to the 3'end) therefore restricting the joining regions that they can recombine with. J4C4 is a pseudo gene and has no protein coding ability and can therefore be excluded. Through the process of pairing the number of possible genes that can be generated is 8.

Why would this be ineffective in fighting pathogens? (1 mark)

This would be ineffective at fighting pathogens because of the limited/small amounts of recombinants that can be generated during recombination producing a small amount of diversity in the immunoglobulin gene. The fundamental reason for this in the murine lambda light chain locus is that the variable genes (V1, V2, and VX) cannot recombines with joining regions upstream restricting the diversity. With there being so many different kinds of infectious diseases with a large amount of variety between them, the immunoglobulin genes need to be equally as diverse to fight off infections.

Describe, with diagrams, two additional ways that diversity of the immunoglobulin lambda light chain genes could be increased. N.B. one of these mechanisms only occurs in the small fraction of lambda light chains that rearrange early in pro-B cells. The names of key proteins should be included. (3 marks)

Junctional diversity

One mechanism that increases diversity in the immunoglobulin genes is the removal and then replacement of nucleotides at hypervariable region three (HV3), this adds additional diversity to the HV3. This diversity is generated during the process of forming a junction between the different genes that are being spliced together to generate the mRNA. The process of P and N nucleotide addition is known as junctional diversity. This junctional diversity is seen in the Pro-B cell primarily on the heavy chains. Figure 1 illustrates this P and N nucleotide addition and the proteins involved during the joining of a V and J region of an immunoglobulin heavy chain gene. Figure 2 shows how TdT generates additional diversity by adding N-nucleotidesfigure 4-08 part 1 of 2figure 4-08 part 2 of 2

Figure.2 The generation of junctional diversity with more detail in the action of RAG, Ku70 Ku80 and TdT on individual nucleotides in the process of N and P nucleotide addition

Combinatorial joining of L and H Chains

Once additional diversity is generated in the heavy and light chains the two combine in the Pre B-cell stage giving the B-cell a suurrgote light chain . The B-cells then proliferate and undergo rearrangment on the light chain developing into the immature niave B cell which later develope into mature B cells.

Describe the four ways in which V(D)J recombination is thought to trigger cancer - 1 paragraph for each (4 marks)

Cryptic recombination

In the antigen receptor loci there are around 3000 RSS, there are also 10 million cryptic RSS in the human genome which are very similar in to the ones found in the antigen loci. When the RAGS bind to the RSS they accidently bind to the cryptic RSS which could be located next to an oncogene and bring the oncogene into the antigen loci. With this oncogene being regulated by strong B and T-cell enhancers this will lead to massive expression of the cancer gene. An example of one of these cryptic RSS being recognised by RAGS and being translocated into the TCR β locus. Cryptic recombination has a low frequency to cause cancer due to many of the cryptic RSS being heavily mutated away from normal RSS not allowing RAGS to bind very well. 2

End donation

This is caused when there are two broken ends of DNA one from the cutting of RAG and the other form elsewhere in the genome possibly an oncogene join together. An example of this is in the BCL-2 gene which is involved with apoptosis and IgH locus, the two translocate together and give rise to the most common translocation in human cancers and is found in the majority of follicular lymphomas. The reason behind the BCL-2 locus being so significant is that the break points in the locus are clustered together(~120bp) even though the locus is quite large, this clustering forms an unusual DNA structure that RAGS can bind to and cut leading to recombination into the heavy chain locus.2

Transposition

The intervening pieces of DNA that are cleaved out during VDJ recombination to bring together different genes were thought to transposes to different parts of the genome. If these were to transpose next to an oncogene they could cause expression of an oncogene. However it has not been show to happen invivo and there are no documented cases of it causing cancer. This was thought to be the cases because of homology between RAGS and RSS and the transposition of DNA therefore allowing the excised pieces of DNA during VDJ recombination to integrate back into the genome.2

Re-integration

The excises piece of DNA can reintegrate back into the genome at another RSS catalysed by RAG proteins, if this happens next to an oncogene it could lead to its expression. It has been calculated that reintegration happens ~5000 times per day in the body. It has not been documented as a major cause of cancer however it is hard to detect. Many (30%) T-cell leukaemia's have no translocations and are believed to be caused by this mechanism.2

Describe another mechanism later in B cell development by which antigen receptor diversity can be enhanced. Include the names of the key proteins (2 marks)

Somatic Hypermutation (SHM)

Somatic hypermutation is a random process that occurs the variable region of the immunoglobulin gene, it predominately occurs in the hypervarible regions in order to prevent mutation in crutial genes which could lead to cell death. In order for it to occur transcription must go through the VDJ region which allows the protein AID (Activation-induced cytidine deaminase) to have accesses to the area which converts cytidine into uracil. Uracil-DNA-glycosylase (UNG) then removes uracil leaving behind the sugar backbone of the DNA. Apurinic/apyrimidinic endonuclease (APE1) then removes the sugar backbone to create a single stranded nick in the DNA. There are three possible processes that can happen to the converted cytidine to uracil, the fist one is that the uracil can be left in the DNA and replication over the DNA can produce a transition mutation (U-G = U-A and C-G). The second is that the uracil can be removed along with the sugar backbone and then translesion synthesis (TLS) polymerase inserts a base at random opposite from where the uracil was removed creating transverstion mutations. The third way is that once the uracil and sugar backbone is removed the mismatch repair enzymes repair the DNA however it is done so incorrectly leading to transverstion mutations.

Class switch recombination requires the same proteins however double stranded breaks occur in the DNA rather than single stranded breaks in SHM. This is thought to be the case due to the being more transcription and AID in the areas undergoing class switch recombination.

Describe the evidence that proteins involved in this process trigger cancer. (3 marks)

A frequent translocation during CSR and SHM that causes cancer in mature B-cells is the Myc-oncogene is translocated into the switch region of the heavy chain.

Evidence that AID is responsible for this has been shown by using two different mice models, one was injected with pristane and the other was iron six deficient both caused a high level of translocations during CSR. Mice that are lacking AID (AID−/−IL6tg) do not show translocations, evidence that AID is responsible1,3,4.

Overexpression of AID has also been shown to increase the frequency of translocations during CSR4. If AID is over expressed in AID -/- spleen B-cells the frequency of c-myc/IgH translocations increases4. The translocation was also found to be dependent of UGA4.

During SHM AID induces mutation into the HV3 however it has also been found that AID is introducing mutation into the uncogenes and activating them. AID has the ability to induce mutation in the HVR but by doing so can induce mutation into uncogenes progressing to cancer 1

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