- Immunoglobulin Genes cont.

Reminder - Major contribution to diversity of Ig comes from recombinatorial diversity.

Bottom line: Over 10 million gene combinations possible ------> 10 million specificities can be generated by this mechanism alone. Actual molecular mechanisms for Ig gene recombination have been the focus of intensive research, and a great deal is now known.

Heptamer - Nonamer and 12/23 Rule [ One Turn-Two Turn Rule]
Recombination Signal Sequences (RSSs)
RSSs consist of a Heptamer (7 nucleotide sequence) separated by either 12 base pairs or 23 base pairs from a nonamer (9 nucleotide sequence). Rule that governs joining is a sequence with one type of spacing (i.e. 12 base pairs) can be joined only to a consensus sequence with the other type of spacing (23 base pairs).
Leroy Hood made the observation that the 12 base pair sequence corresponded to approximately 1 turn of the DNA helix, while 23 base pairs corresponded to approximately 2 turns of the DNA helix ------ thus, one turn/two turn rule.

- example of heptamer

example of nonamer:

For example, In Mouse Kappa light chain:

Each Vk gene segment is followed by a heptamer / nonamer sequence with 12 base pair spacing.

Each Jk gene segment is preceeded by a consensus segment with 23 base pair spacing.
[ Interestingly, Lamda is just reversed (The V RSS has 23 bp spacer and the J’s have RSS with 12 bp spacing) ]

This rule serves to prevent one V segment from recombining with another V segment.

Heavy Chains
VH followed by 23 base pair type RSS
DH flanked on either side by the 12 base pair type RSS
JH preceeded by 23 base pair type RSS.
V is prevented from rearranging directly with J because they have the same spacing.
Recombination involves breakage and reunion of the DNA. Mechanisms for Ig gene rearrangement are summarized in text .

Interesting point: There is a connection between joining of the V and C segments and the activation of transcription. Unrearranged genes are not transcribed. This is believed to be because rearrangement brings promoter regions (upstream from each of the V region sequences) into closer proximity to enhancer sequences (upstream from the C region gene segments).

Allelic Exclusion
A single productive rearrangement of VL-JL and of VH-DH -JH occurs in a given lymphocyte. The event involves the genes of only one of the homologous chromosomes, the alleles on the other chromosome are not expressed in the same cell. Most likely, the cell tries to recombine gene segments until a productive rearrangement is achieved. Allelic exclusion is caused by the suppression of further rearrangement as soon as the active light or heavy chain is produced. It has been estimated that ~ 2/3 rearrangements are non-productive.
Light and heavy chain genes rearrange independently in the developing B lymphocyte. The cell rearranges and synthesizes H chain first, the successful synthesis of a heavy chain triggers light chain rearrangement.
Kappa chain genes rearrange first and if rearrangement fails on both homologous chromosomes, then lambda chain genes can attempt to rearrange. This is why in both humans and mice, the majority of light chains are kappa light chains.

Ultimately Immunoglobulin diversity is generated in many different ways:

Recombinatorial Diversity: Due to the multiplicity of gene segments in the germline (i.e. multiple V’s, D’s, J’s in the heavy chain gene array) coupled with the rearrangement of these gene segments during B lymphocyte development.

Rearrangement based on complementary base pairing between RSS sequences.

The recognition of the RSS sequences and the rearrangement which occurs is mediated by RAG-1 and RAG-2 gene products. (RAG = recombinase activating gene)

Ig gene rearrangment only occurs when both the RAG-1 and RAG-2 genes are expressed within the B lymphocyte.

Junctional Diversity:There is variation in the position at which V-J light chain gene segments join and V-DJ and D-J heavy chain gene segments join. These "imprecise" junctions help to form the CDR3 of both the light and heavy chain V regions. For instance, there is a lot of amino acid variability at position 96 of the kappa light chain due to slight differences at the precise joining between V and J. Different codons result. Position 96 is right in the CDR3 of the variable region. A process known as N-region Diversity (N-nucleotide addition) also contributes to this phenomenon. A few nucleotides (up to 15) may be inserted between DH and JH and between VH and DH by means of an enzyme known as, terminal deoxynucleotidyl transferase (tDt). The synthesis of these nucleotides is without the use of a template. In addition, a second mechanism known as P-nucleotide addition can result in the addition of short palindromic (p= palindromic) sequences at these coding junctions following DNA strand breakage.

To top it off, an as yet unknown exonuclease can also remove nucleotides (nucleotide deletion) from the segments prior to ligation.

Combinatorial Diversity:H and L chains are encoded on separate chromosomes and therefore are synthesized separately. As a consequence, they enter the rough endoplasmic reticulum independently. Chaperone proteins present within the RER are necessary to hold and retain the H and/or light chains until they can join together. The two chains must ultimately come together to form an intact Ig with a unique antigen-binding capacity. Therefore, you have the random possible combinations of all possible Hs and Ls.

Somatic Hypermutation:Point mutations (substitutions of individual nucleotide pairs) occur at a high frequency within rearranged V genes. Within the V region the somatic mutation rate is 10^-3 per base pair per generation, which is a million times greater than normal!

This process leads to affinity maturation of the antibody response. Somatic mutation is known to occur in B lymphocytes positioned in the germinal centers of the secondary lymphoid organs and requires the presence of Th lymphocytes and Th cytokines. Nude mice (congenitally athymic) do not have T cells and therefore do not have germinal centers, and do not show affinity maturation.

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