What is combinatorial diversity

These features have provoked speculation that the RAG complex originated as a transposase whose function was adapted by vertebrates to allow V gene segment recombination, thus leading to the advent of the vertebrate adaptive immune system. Consistent with this idea, no genes homologous to the RAG genes have been found in nonvertebrates. The in vivo roles of the enzymes involved in V D J recombination have been established through natural or artificially induced mutations.

Mice in which either of the RAG genes is knocked out suffer a complete block in lymphocyte development at the gene rearrangement stage.

Mice lacking TdT do not add extra nucleotides to the joints between gene segments. A mutation that was discovered some time ago results in mice that make only trivial amounts of immunoglobulins or T-cell receptors. Such mice suffer from a s evere c ombined i mmune d eficiency—hence the name scid for this mutation.

These mice have subsequently been found to have a mutation in the enzyme DNA-PK that prevents the efficient rejoining of DNA at gene segment junctions.

Mutations of other proteins that are involved in DNA joining also give the scid phenotype. Antibody diversity is generated in four main ways. Two of these are consequences of the recombination process just discussed see Sections and which creates complete immunoglobulin V-region exons during early B-cell development. The third is due to the different possible combinations of a heavy and a light chain in the complete immunoglobulin molecule.

The fourth is a mutational process that occurs in mature B cells, acting only on rearranged DNA encoding the V regions. The gene rearrangement that combines two or three gene segments to form a complete V-region exon generates diversity in two ways. First, there are multiple different copies of each type of gene segment, and different combinations of gene segments can be used in different rearrangement events.

This combinatorial diversity is responsible for a substantial part of the diversity of the heavy- and light-chain V regions. Second, junctional diversity is introduced at the joints between the different gene segments as a result of addition and subtraction of nucleotides by the recombination process. A third source of diversity is also combinatorial, arising from the many possible different combinations of heavy- and light-chain V regions that pair to form the antigen-binding site in the immunoglobulin molecule.

The two means of generating combinatorial diversity alone could give rise, in theory, to approximately 3. Coupled with junctional diversity, it is estimated that as many as 10 11 different receptors could make up the repertoire of receptors expressed by naive B cells. Finally, somatic hypermutation introduces point mutations into the rearranged V-region genes of activated B cells, creating further diversity that can be selected for enhanced binding to antigen.

We will discuss these mechanisms at greater length in the following sections. There are multiple copies of the V, D, and J gene segments , each of which is capable of contributing to an immunoglobulin V region. Many different V regions can therefore be made by selecting different combinations of these segments. So, in all, different light chains can be made as a result of combining different light-chain gene segments.

During B-cell development, rearrangement at the heavy-chain gene locus to produce any one of the possible heavy chains is followed by several rounds of cell division before light-chain gene rearrangement takes place.

The particular combination of gene segments used to produce a heavy chain does not appear to restrict the choice of gene segments that can be recombined to assemble a light-chain variable region. Thus, in theory any one possible heavy chain can be produced together with any one possible light chain in a single B cell. As both the heavy- and the light-chain V regions contribute to antibody specificity , each of the different light chains could be combined with each of the approximately 11, heavy chains to give around 3.

This theoretical estimate of combinatorial diversity is based on the number of germline V gene segments contributing to functional antibodies see Fig. In practice, combinatorial diversity is likely to be less than one might expect from the theoretical calculations above.

One reason for this is that not all V gene segments are used at the same frequency; some are common in antibodies, while others are found only rarely. It is also clear that not every heavy chain can pair with every light chain ; certain combinations of V H and V L regions result in failure to assemble a stable immunoglobulin molecule.

Cells that have heavy and light chains that cannot pair may continue to undergo light-chain gene rearrangement until a suitable light chain is produced, or may be eliminated, but in both cases a heavy- and light-chain combination that does not pair is lost from the repertoire.

Nevertheless, it is thought that most heavy and light chains can pair with each other, and that this type of combinatorial diversity has a major role in the formation of an immunoglobulin repertoire with a wide range of specificities.

In addition, two further processes add greatly to repertoire diversity—imprecise joining of V, D, and J gene segments and somatic hypermutation. Of the three hypervariable loops in the protein chains of immunoglobulins , two are encoded within the V gene segment DNA. In both heavy and light chains, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides at two steps in the formation of the junctions between gene segments.

The added nucleotides are known as P-nucleotides and N-nucleotides and their addition is illustrated in Fig. The introduction of P- and N-nucleotides at the joints between gene segments during immunoglobulin gene rearrangement. After more P-nucleotides are so called because they make up palindromic sequences added to the ends of the gene segments.

After the formation of the DNA hairpins as described in Section , the RAG protein complex catalyzes a single-stranded cleavage at a random point within the coding sequence but near the original point at which the hairpin was first formed. When this cleavage occurs at a different point from the initial break, a single-stranded tail is formed from a few nucleotides of the coding sequence plus the complementary nucleotides from the other DNA strand see Fig.

In most light-chain gene rearrangements, DNA repair enzymes then fill in complementary nucleotides on the single-stranded tails which would leave short palindromic sequences at the joint, if the ends are rejoined without any further exonuclease activity see below.

In heavy-chain gene rearrangements and in some human light-chain genes, however, N-nucleotides are first added by a quite different mechanism. N-nucleotides are so called because they are nontemplate-encoded. They are added by the enzyme terminal deoxynucleotidyl transferase TdT to single-stranded ends of the coding DNA after hairpin cleavage.

After the addition of up to 20 nucleotides by this enzyme, the two single-stranded stretches at the ends of the gene segments form base pairs over a short region.

Repair enzymes then trim off any nonmatching bases, synthesize complementary bases to fill in the remaining single-stranded DNA, and ligate it to the P-nucleotides see Fig. N-nucleotides are found especially in the V-D and D-J junctions of the assembled heavy-chain gene; they are less common in light-chain genes because TdT is expressed for only a short period in B-cell development, during the assembly of the heavy-chain gene, which occurs before that of the light-chain gene.

Nucleotides can also be deleted at gene segment junctions. This is accomplished by as yet unidentified exonucleases. Thus, the length of heavy-chain CDR3 can be even shorter than the smallest D segment.

In some instances it is difficult, if not impossible, to recognize the D segment that contributed to CDR3 formation because of the excision of most of its nucleotides. Deletions may also erase the traces of P-nucleotide palindromes introduced at the time of hairpin opening. For this reason, many completed V D J joins do not show obvious evidence of P-nucleotides. As the total number of nucleotides added by these processes is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint.

Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements. As roughly two in every three rearrangements will be nonproductive, many B cells never succeed in producing functional immunoglobulin molecules, and junctional diversity is therefore achieved only at the expense of considerable wastage.

We will discuss this further in Chapter 7. The mechanisms for generating diversity described so far all take place during the rearrangement of gene segments in the initial development of B cells in the central lymphoid organs. There is an additional mechanism that generates diversity throughout the V region and that operates on B cells in peripheral lymphoid organs after functional immunoglobulin genes have been assembled. This process, known as somatic hypermutation, introduces point mutations into the V regions of the rearranged heavy- and light-chain genes at a very high rate, giving rise to mutant B-cell receptors on the surface of the B cells Fig.

Some of the mutant immunoglobulin molecules bind antigen better than the original B-cell receptors, and B cells expressing them are preferentially selected to mature into antibody -secreting cells. This gives rise to a phenomenon called affinity maturation of the antibody population, which we will discuss in more detail in Chapters 9 and Somatic hypermutation introduces variation into the rearranged immunoglobulin variable region that is subject to negative and positive selection to yield improved antigen binding.

In some circumstances it is possible to follow the process of somatic hypermutation more Somatic hypermutation occurs when B cells respond to antigen along with signals from activated T cells. The immunoglobulin C-region gene, and other genes expressed in the B cell , are not affected, whereas the rearranged V H and V L genes are mutated even if they are nonproductive rearrangements and are not expressed. The pattern of nucleotide base changes in nonproductive V-region genes illustrates the result of somatic hypermutation without selection for enhanced binding to antigen.

The pattern of base changes in the V regions of expressed immunoglobulin genes is different. Mutations that alter amino acid sequences in the conserved framework regions will tend to disrupt basic antibody structure and are selected against. In contrast, the result of selection for enhanced binding to antigen is that base changes that alter amino acid sequences, and thus protein structure, tend to be clustered in the CDRs, whereas silent mutations that preserve amino acid sequence and do not alter protein structure are scattered throughout the V region.

The mechanism of somatic hypermutation is poorly defined, but there have been several new discoveries that shed some light. It is known that mutation requires the presence of enhancers , DNA sequences that enhance the trans-cription of immunoglobulin genes in B cells, as well as a transcriptional promoter. The promoter, and the sequences that are the target of mutation need not derive from immunoglobulin V genes, however.

The generation of new mutations in V regions in mutating B cells has recently been shown to be accompanied by double-stranded breaks in the DNA which are thought to then be repaired in an error-prone way. In addition, it has recently been discovered that deficiency in an RNA editing enzyme called Activation Induced Cytidine Deaminase, blocks the accumulation of somatic hypermutations.

The mechanism by which this enzyme contributes to hypermutation is unknown. Interestingly, deficiency of this enzyme also abrogates the rearrangement of C-region genes that underlies the immunoglobulin class switching seen in activated B cells see Section As we have seen in the preceding sections, a proportion of the immunoglobulin diversity in an adult human derives from the existence of a variety of germline gene segments , and a proportion from somatic alterations acquired during the lifetime of the individual.

This particular combination of heritable and acquired components of diversity operates in several mammalian immune systems, including those of humans and mice. Other species achieve a mix of inherited and acquired diversity by different means.

Overall, it would appear that there is strong selective pressure to generate sufficient diversity in the immune system to protect the organism from common pathogens, and several different mechanisms have evolved toward this end. In birds, rabbits, cows, pigs, sheep, and horses there is little or no germline diversity in the V, D, and J gene segments that are rearranged to form the genes for the initial B-cell receptors, and the rearranged V-region sequences are identical or similar in most immature B cells.

These B cells then migrate to specialized microenvironments, the best known of which is the bursa of Fabricius in chickens. Here, B cells proliferate rapidly, and their rearranged immunoglobulin genes undergo further diversification. In birds and rabbits this occurs by a process that includes gene conversion , in which an upstream V segment pseudogene exchanges short sequences with the expressed rearranged V-region gene Fig.

In sheep and cows, diversification is the result of somatic hypermutation, which occurs in an organ known as the ileal Peyer's patch. Somatic hypermutation probably also contributes to immunoglobulin diversification in birds and rabbits. The diversification of chicken immunoglobulins occurs through gene conversion. In chickens, all B cells express the same surface immunoglobulin slg initially; there is only one active V, D, and J gene segment for the chicken heavy-chain gene and one more Diversity within the immunoglobulin repertoire is achieved by several means.

Perhaps the most important factor that enables this extraordinary diversity is that V regions are encoded by separate gene segments , which are brought together by somatic recombination to make a complete V-region gene. Many different V-region gene segments are present in the genome of an individual, and thus provide a heritable source of diversity. Additional diversity, termed combinatorial diversity , results from the random recombination of separate V, D, and J gene segments to form a complete V-region exon.

Variability at the joints between segments is increased by the insertion of random numbers of P- and N-nucleotides and by variable deletion of nucleotides at the ends of some coding sequences. The association of different light- and heavy-chain V regions to form the antigen-binding site of an immunoglobulin molecule contributes further diversity.

Finally, after an immunoglobulin has been expressed, the coding sequences for its V regions are modified by somatic hypermutation upon stimulation of the B cell by antigen. The combination of all these sources of diversity generates a vast repertoire of antibody specificities from a relatively limited number of genes.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed. Turn recording back on. National Center for Biotechnology Information , U. New York: Garland Science ; Search term. The generation of diversity in immunoglobulins. Immunoglobulin genes are rearranged in antibody-producing cells In nonlymphoid cells, the gene segments encoding the greater part of the V region of an immunoglobulin chain are some considerable distance away from the sequence encoding the C region.

Figure 4. The assembled variable genes can be revised and edited resulting in a change of their affinity and even specificity. Due to somatic hypermutation, the affinity of synthesized antibody increases even more.

Another variant of combinatorial recombination is joining of complete variable genes with one of the several constant genes and the formation of various immunoglobulin isotypes with different effector functions but with the same antibody specificity. Consequently, these processes not only develop the antibody repertoire but also solve some other problems of the adaptive immune response. Abstract Combinatorial association of immunoglobulin gene elements is the most important process in the creation of extreme diversity of antibody molecules.