- Immunoglobulin Genes

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 of immunoglobulin.

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 Mouse Myeloma cell DNA and compared it to the light chain gene organization present in mouse embryonic DNA. For this analysis they utilized restriction enzymes to cut DNA into fragments which were then analyzed by the technique known as Southern Blotting.

Finding: In differentiated cells able to produce antibodies (i.e. myeloma cells and B lymphocytes) individual V and C gene segments are brought together to form a combined 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 (as well as many other mammals). These recombination events are the only known form of site specific DNA rearrangement in vertebrates.

Quote from Philip Leder (another 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."
 

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-V_k1---L-V_k2------L-V_kn-------J_k1-J_k2-J_k3-J_k4-J_k5-----C_k--3'

• V-J joining forms the coding sequence for the k 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 Light chain.
• The J chain codes for last 13 amino acids in the variable region of light chain.

In addition, there are leader sequences just before each V gene segment. The leader sequences are transcribed and translated and are necessary for directing the nascent polypeptide into the RER during protein synthesis.  The leader sequences are then removed once the synthesis of the polypeptide is completed.

To Recap:
During B lymphocyte development, site specific DNA rearrangement occurs. One V and one J gene segment come together 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 and subsequent synthesis of a primary transcript, RNA splicing removes introns joining V J to C to produce a functional mRNA.

Remember:
introns - intervening sequences removed by RNA splicing
exons - sequences which remain and are joined to form mRNA
 
Heavy Chain Genes

In the embryonic (germline configuration) the D segments lie  between V and J gene segments.
Presence of D segments was first proposed by Leroy Hood (presently of University of Washington, Seattle - big player in human genome project)

During Heavy Chain Gene Rearrangment, 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 Mouse Heavy Chain genes]

5'--L-V_H1--L-V_H2-----L-V_Hn---//--D_H1-D_H2----D_H12---//J_H1-J_H2-J_H3-J_H4----//--Cm-----Cd------Cg3-----Cg1----Cg2b----Cg2a---Ce---Ca--3'

C segments correlate with the different heavy chain isotypes (or 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.

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.

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