Genetics of Antibody Diversity
Dogma in biology for many years - 1 gene, 1 polypeptide, entrenched in the mind set of investigators.
The enigma of the variable and constant regions of the heavy and light chain puzzled people tremendously.
In 1965 - Dreyer and Bennett proposed that there must be at least two genes for one polypeptide chain in antibody formation. They proposed that there must be 100s or even thousands of different genes for the V regions but only 1 C region gene for each isotype.
In 1976, Tonegawa and Hozumi were the first to provide the experimental proof of the Dreyer and Bennett hypothesis.
Tonegawa and Hozumi analyzed the light chain genes from Myeloma cell DNA and compared the organization to that of light chain genes present in Whole mouse embryonic DNA.
Finding: In differentiated cells able to produce antibodies (i.e. myeloma cells and B lymphocytes) the V and C gene segments are brought together to form a V-C segment.
In other words, the Ig genes undergo gene rearrangement during B cell development. Ig gene rearrangement has been extensively studied and is known to be the primary mechanism for the generation of Ig diversity in humans and mice. These recombination events are the only known form of site specific DNA rearrangement in vertebrates.
Quote from Philip Leder (an important investigator in this field of research).
"The genes ultimately specifying the structure of each antibody are not present as such in germ cells, or in the cells of the early embryo. Rather than harboring a set of complete and active antibody genes, these cells contain bits and pieces of the genes; a kit of components.
The components are shuffled in the cells of the immune system called B lymphocytes as those cells develop and mature. The shuffling can lead to a different result in each of millions of lines of cells. Individual mutations amplify the diversity. The result is that in the mature descendents of each line, a unique gene is assebled, whose information is expressed in the form of unique antibody."
Example of Kappa Light Chain of Mouse
~300 V (variable) gene segments
one C segment
five J (joining) segments (one of these five is a non-functional pseudogene).
During B cell development, one V region segment is rearranged to join with one J region segment.
In the germline DNA, the kappa light chain genes are arranged as follows:
5'---L-Vk1---L-Vk2------L-Vkn-------Jk1-Jk2-Jk3-Jk4-Jk5-----Ck--3'
V-J joining forms the light chain variable region.
J segments are an extra set of gene segments separated in the genome from V segments.
The V segments and J segments are joined to form the functional V region of the L chain gene.
The J chain codes for last 13 amino acids in the variable region.
In addition, there are leader sequences just before each VL gene segment. The leader sequences are transcribed and translated but are excised from the protein before it is secreted.
During B lymphocyte development, DNA rearrangement occurs. One VL and one JL gene segment come together in the genome and the intervening sequences are deleted. All other cells of the body have the entire set of Ig genes. Only B lymphocytes rearrange them.
Following gene rearrangement, RNA splicing removes introns joining VLJL to C to produce the functional mRNA.
Remember:
introns - intervening sequences removed by RNA splicing
exons - sequences which remain to form mRNA
Heavy Chains
Tandem array of VH gene segments (300-1000) in mice
Array of JH gene segments (4)
Also DH segmetns (D for diversity) 12
The D segments lie in germ line between V and J.
Presence of D segments was first proposed by Leroy Hood (presently of University of Washington, Seattle)
V-D-J joining must occur and it occurs in two stages:
1) recombination of one D segment with one J segment
2) recombination of a V segment with the DJ segment
Diagrams: [germline configuration of Heavy Chain genes]
5'--L-VH1--L-VH2-----L-VHn---//--DH1-DH2----DH12---//JH1-JH2-JH3-JH4------//--Cm--------Cd---------Cg3--------Cg1-------Cg2b------Cg2a-------Ce-------Ca-------3'
C segments correlate with the different isotypes (and subclasses of Ig) With this variety of different gene segments, the rearrangement and joining results in a unique DNA sequence for each combination of segments.
Bottom line: Over 10 million gene combinations possible ------> 10 million specificities.
Major contribution to diversity of Ig comes from this recombinatorial diversity. Actual molecular mechanisms for recombination are not fully understood but important signal sequences are known.
Heptamer - Nonamer and 12/23 Rule [ One Turn-Two Turn Rule]
Recombination Signal Sequences (RSSs)
The 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: C A C A G T G
example of nonamer: A C A A A A A C C
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.
Lamda is just reversed (V has 23 bp and J has 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 but you will not be responsible for the mechanisms and we will not cover in class.
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.
Light and heavy chains independently. Cell rearranges and synthesizes H chain first, success 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.
Immunoglobulin diversity is generated in many different ways:
Recombinatorial Diversity
V-J
V-D-J
Rearrangement based on complementary base pairing between RSS sequences mediated by RAG-1 and RAG-2 gene products.
heptamer 12 nonamer can only base pair with heptamer 23 nonamer.
Junctional Diversity
There is variation in the position at which V-J light chain gene segments join and D-J heavy chain gene segments join. These "imprecise" junctions help to form the CDR3 of both the light and variable chains. For instance, there is a lot of amino acid variability at position 96 of the kappa light chain due to slight differentces at the precise joining between V and J. Different codons result. Position 96 is right in the CDR3 of the variable region. Book describes molecular model for this. Consequence of enzymatic mechanisms involved in breaking and rejoining DNA.
N-region Diversity
A few nucleotides (up to 15) may be inserted between DH and JH and between VH and DH by means of the enzyme, terminal deoxynucleotidyl transferase (tDt), without the use of a template.
Combinatorial Diversity
H and L chains are synthesized separately and therefore enter the endoplasmic reticulum independently. The two chains come together to yield a certain antigen-binding capacity. Therefore, you have random possible combinations of all possible Hs and Ls.
Somatic Mutation
Point mutations (substitutions of individual nucleotide pairs) occur particularly in the variable regions of 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. Leads to affinity maturation of the antibody response. Somatic mutation is known to occur in the germinal centers of the secondary lymphoid organs and requires the presence of Th lymphocytes. Nude mice (congenitally athymic) do not have T cells and therefore do not have germinal centers, and do not show affinity maturation.
Recombination Activating Genes [RAG-1 and RAG-2]
Site specific recombinases are required for immunoglobulin gene rearrangement. So far, two genes have been implicated as coding for recombinases: RAG-1 and RAG-2.
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