ADVERTISEMENTS:
In this article we will discuss about the organisation of immunoglobulin genes with special reference to multi-gene organisation.
It has become clear that the B-lymphocytes of the immune system produce antibodies in the presence of antigen. The antibodies are formed from the assembly of some protein chains. In mammals, enormous amount of antibodies are produced. For this, there must be millions of genes for each antibody.
But how can it be possible because a mammalian genome does not contain more than about a million of genes, out of which only a fraction of genome directs the synthesis of antibodies. This clearly shows that neither the germ cells nor embryonic cells contain a complete set of all genes but have the basic genes which are shuffled during developmental stages of B-lymphocytes.
In germ line DNA, multiple gene segments encode a single immunoglobulin heavy or light chain. These gene segments are carried in the germ cells that cannot be transcribed and translated into heavy and light chains until these are arranged into functional genes. During the differentiation of B-cells in bone marrow these gene segments are randomly shuffled about 108 specificities by a dynamic genetic system.
This is maintained by germ line theory. Differentiation of B-cells from a progenitor B-cell to a mature cell involves an ordered progress in rearrangement of immunoglobin genes. When the process of B-cell division is over, a mature immuno-competent B-cell contains a single functional variable region DNA sequence for its heavy chain and a single functional variable region DNA sequence for its light chain.
The somatic variation theories maintained that the genome contains relatively a small number of Ig genes which generate a large number of antibody specificity in the somatic cells through the mechanism of either recombination or mutation. Still these two theories could not explain how the stability be maintained in the C-region while some diversifying mechanism is involved to degenerate the V-region.
This has been confirmed through the recent evidences that a single variable region sequence specific for a particular antigen can be associated with the multiple sequences of C-region of heavy chains. It means that the different isotypes of antibodies (i.e. IgG, IgM) can be expressed having identical sequences of V-region.
(i) Dryer and Bennett’s Two Gene Model:
ADVERTISEMENTS:
In 1965, W.Dryer and J. Bennett in their classical theoretical paper suggested for encoding of immunoglobulin chains. The two separate genes encode two different chains, one the light chain and the other heavy chain. They hypothesized that the two genes must come together and form a complete set of genes that can transcribe and translate the full message and can yield a single heavy or light protein chain.
This hypothesis predicted the well established theory of one-gene-one polypeptide hypothesis of Beadle and Tatum for that they were awarded Nobel Prize in 1958 with J. Lederberg. However, they could not provide the experimental data; it was merely a theoretical framework. After the development of technologies this hypothesis lent support to disclose the mystery of gene organization at molecular level.
Experimental evidence of gene rearrangement:
For the first time, Hozumi and Tonegawa (1976) provided the experimental evidence for the rearrangement of two separate genes encoding the V and C-regions of immunoglobulin during the course of differentiation of B-lymphocytes, and produce millions of antibodies. For this novel work, Tonegawa was awarded Nobel Prize in 1987 in medicine and physiology.
They used the newly developed Southern Blotting Technique. They took myeloma cells because they are like Plasma cells and produce large amount of single antibodies, and prepared radiolabelled RNA i.e. 32p-mRNA for K-light and heavy chains, and also for constant chain. 32P- mRNA was used as probe to test two kinds of cells, embryonic cells (that do not produce antibodies) and B-cells (produces antibodies) (Fig. 22.15)
The DNA of both myeloma cells and embryonic cells was treated with restriction enzymes and subjected to gel electrophoresis. The gel was then sliced; DNA fragments were eluted from the slice, denatured into single stranded DNA and finally incubated with 32p-mRNA encoding K- light chain.
The 32p-mRNA probe hybridised with two bands from the germ line embryonic DNA, but with only a single band from the differentiated myeloma DNA. This clearly reveals that in the fully differentiated plasma cells, which is represented by the myeloma cells, the genes for V and C regions had gone rearrangement. Now they are present together on a single restriction DNA fragment, that is why the 32P-mRNA probe hybridized with a single band only.
(ii) Multi-Gene Organization of Immunoglobulin Gene:
The result of Hozumi and Tonegawa (1976) are analogous to the theoretical two gene model of Dryer and Bennett (1965). This provides evidence for organization of multi-gene family into the immunoglobulin gene. In the embryonic cells the DNA encoding C-regions is far away from the DNA that encodes for V-region.
ADVERTISEMENTS:
In plasma cells (i.e. cells producing antibodies, also B-cells) and C and V-regions are together (Fig. 22.16). The k and λ light chains and the heavy chains are encoded by separate multi-gene families situated on different chromosome, that contain a series of coding sequences which are known as gene segments. The k and λ light chain families contain L, V, J and C gene segments, whereas the heavy chain family contains L, V, D, S and C gene segments.
Fig. 22.16 : Arrangement of gene segments of λ-light chain in embryonic and myeloma DNA molecules. RE, restriction enzyme.
During the process of differentiation of B-cells, a long segment of DNA is deleted resulting in close rearrangement of V (i.e. V2 segment to J-segment). The RNA transcript of immunoglobulin gene that contains intron (non-coding segment within the gene) is processed and correct transcript of mRNA is formed which is translated into a polypeptide light chain.
The rearranged VJ-gene segments encode the V-region of the light chain, whereas VDI-gene segments encode the V-region of the heavy chain. The C-segments encode the C-region of the light or heavy chain of the gene segment encodes a short signal sequence.
ADVERTISEMENTS:
The signal sequence guides the light or heavy chain through endoplasmic reticulum but is broken before assembly of the immunoglobulin molecule. Therefore, the amino acids that correspond to L-gene segment do not appear in light or heavy chains.
Multi-gene families of λ-chain, K-chain and heavy chain:
For the first time Tonegawa (1983) gave the evidence that V-region of light chain is encoded by two gene segments (V1 and V2) by closing the germ-line gene encoding V-region of mouse λ light chain. The complete sequence of nucleotide was determined.
When it was compared with known sequences of the λ-chain V-region, a discrepancy was found. In mouse λ-chain multi-gene family contains two V gene segment (V1 and V2), four J-gene segment (J1, J2, J3 and J4) and four C gene segment (C1, C2, C3, C4). The arrangement of the gene segments is shown in Fig. 22.17A.
ADVERTISEMENTS:
The gene segments, J4 and C4, are defective, therefore, called pseudogenes. A functional V- region of λ-chain gene consists of two coding segments i.e. exons (a V-gene segment and a J- segment) which are separated by a non-coding sequence (i.e. intron) in un-rearranged germ line DNA. In humans, there are an estimated 100 V-gene segments, 6 J-segment and 6 C-segments.
In mouse, the k-chain gene family consists of about 300 V gene segments, five J- segment (one segment is pseudo gene) and a single C-gene segment. Unlike λ chain there are no sub classes of k-light chain as only one C-gene segment is found (Fig. 22.17 B). In humans, the k-chain gene family consists of about 100 V-gene segments, 5J segments and a single C-segment.
Fig. 22.17 : Germ line organization of λ-light chain (A) , к-light chain (B), and heavy chain (C) gene segment in the mouse.
In mouse the heavy chain multi-gene family of immunoglobulin is like λ and k chain but a little complex. It is located on chromosome 12. The heavy chain multi-gene family consists of about 200-1000 V-gene segments, 13 D-gene segments, four J-gene segments and a series of C-gene segments (Fig. 22.17C).
Each V-gene segment has a leader sequence a short distance upstream from it. The J-gene segments are located downstream from the D-gene segment. Each C-gene segment encodes the C-region of an immunoglobulin heavy chain iso-type. Similarly each C-gene segment encodes separate domain of the heavy chain C-region. In mouse C-gene segments are arranged in the order Cµ – Cδ – CƳ3 – CƳ1 – CƳ2b – CƳ2a – Cɛ – Cα.
