The Immunology of the Generation of Diversity -- germline theory and somatic cell theory

How the genes which are necessary to produce a vast number - probably more than a hundred million - of different forms of antibodies are inherited from generation to generation, and by what mechanism large numbers of genes in an individual somatic body are maintained have been themes among immunologists for a long time. It has been argued for a long time how so-called antibody diversity is genetically generated.

There were two theories when I went to the Institute of Immunology in Swizerland and started research on this problem in the begining of the 1970s. The first theory was the so-called "germline theory" in which vast numbers of genes in each individual body are inherited as in from the parents. On the other hand, the second theory supposed that only a limited number of antibody genes are inherited from the parents, but somehow diversity is generated by a genetic mechanism during the process of somatic generation and growth of each individual body.

However, we couldn't really obtain experimental evidence for either theory. Finally in the 1970's, restriction enzyme and DNA recombination techniques were developed. Using these methods, research on the genes which correspond to a specific protein in a complex living creature became possible.

Antibody genes have very special structures. It has become clear by observing antibody genes in germ cells, which do not produce antibodies, that their structure is very different from various known genes. For example, by observing how genes on one light chain are arranged in DNA it was found that the part called the variable region is coded separately in several sequences some distance apart in the DNA. Moreover, it was found that the part that codes the invariable region has its own DNA sequence separated from the variable region.

On the other hand, if one observes the genes in cells that produce antibodies, namely B cells or B lymphocytes, in contrast to the case of germ cells, the DNA used for the variable region exists as one continuous sequence. In other words, the V and J gene segments are contiguous (see Fig. 2). Furthermore, in this cell, when the gene is expressed, RNA is produced using this DNA as a template. Then recombination occurs during the process in which the RNA moves from the nucleus to the place where protein is synthesized. By doing so, a sequence not directly related to protein sequence, called an Intron (intervening sequence) is eliminated, messenger RNA is produced, and protein is synthesized on the ribosome (the substance that synthesizes protein).

Recombination of Antibody Gene Fragments

An important point of this discovery is that, unlike the behavior of the genes which has been recognized up to the present, in the case of antibody genes, gene fragments are restructured during the generation and the development of a cell.

When this phenomenon is investigated in more detail, it is more complicated in the case of heavy chains. In the case of heavy chains, instead of two types of gene fragments, the variable region is coded by three different types, called V, D, and J. In this case, in order for this gene to be expressed, one of the many Vs, one of the many Ds, and one of the Js are connected to each other in series. A complete gene which can produce a heavy chain will be created by this process.

What is the significance of the existence of these genes as fragments in germ cells? In Fig.3, in the case of a heavy chain, about 300 V gene fragments, 20 D gene fragments, and 5 J gene fragments exist in germ cells. In the process when a cell is developed and B lymphocytes are generated, one of each type of fragment from the pools of these three gene fragments are selected. A complete gene for a heavy chain can be created by the various combinations. Therefore, it can be simply calculated that, from all the combinations of 300 types of V gene fragments, 20 types of D gene fragments, and 5 types of J gene fragments, 24,000 different types of variable regions can be created. As many as 24,000 different types of genes can be produced from very few, several hundred at most, gene fragments inherited from the parents. The somatic cell theory was proved by this discovery.