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In this lecture educator explains the Organization and Expression of Immunoglobulin Genes – I. The vertebrate immune system is its ability to respond to an apparently limitless array of foreign antigens. As immunoglobulin (Ig) sequence data accumulated, virtually every antibody molecule studied was found to contain a unique amino acid sequence in its variable region but only one of a limited number of invariant sequences in its constant region. The genetic basis for this combination of constancy and tremendous variation in a single protein molecule lies in the organization of the immunoglobulin genes.
Genetic Model Compatible with Ig Structure
- Immunoglobulin genes had to account for the following properties of antibodies:
- The vast diversity of antibody specificities.
- The presence in Ig heavy and light chains of a variable region at the amino-terminal end and a constant region at the carboxyl-terminal end.
- The existence of isotypes with the same antigenic specificity, which result from the association of a given variable region with different heavy-chain constant regions.
Germ-Line and Somatic-Variation Models: For several decades, immunologists sought to imagine a genetic mechanism that could explain the tremendous diversity of antibody structure. Two different sets of theories emerged. The germ-line theories maintained that the genome contributed by the germ cells, egg and sperm, contains a large repertoire of immunoglobulin genes; thus, these theories invoked no special genetic mechanisms to account for antibody diversity. They argued that the immense survival value of the immune system justified the dedication of a significant fraction of the genome to the coding of antibodies. In contrast, the somatic-variation theories maintained that the genome contains a relatively small number of immunoglobulin genes, from which a large number of antibody specificities are generated in the somatic cells by mutation or recombination. Neither the germ-line nor the somatic-variation proponents could offer a reasonable explanation for this central feature of immunoglobulin structure. Germ-line proponents found it difficult to account for an evolutionary mechanism that could generate diversity in the variable part of the many heavy- and light-chain genes while preserving the constant region of each unchanged. Somatic-variation proponents found it difficult to conceive of a mechanism that could diversify the variable region of a single heavy- or light-chain gene in the somatic cells without allowing alteration in the amino acid sequence encoded by the constant region. A third structural feature requiring an explanation emerged when amino acid sequencing of the human myeloma protein called Ti1 revealed that identical variable region sequences were associated with both Ƴ and µ heavy chain constant regions.
Two-Gene Model: In an attempt to develop a genetic model consistent with the known findings about the structure of immunoglobulins, W. Dreyer and J. Bennett suggested, in their classic theoretical paper of 1965, that two separate genes encode a single immunoglobulin heavy or light chain, one gene for the V region (variable region) and the other for the C region (constant region). They suggested that these two genes must somehow come together at the DNA level to form a continuous message that can be transcribed and translated into a single Ig heavy or light chain. Moreover, they proposed that hundreds or thousands of V-region genes were carried in the germ line, whereas only single copies of C-region class and subclass genes need exist. The strength of this type of recombinational model (which combined elements of the germ-line and somatic variation theories) was that it could account for those immunoglobulins in which a single V region was combined with various C regions. By postulating a single constant region gene for each immunoglobulin class and subclass, the model also could account for the conservation of necessary biological effector functions while allowing for evolutionary diversification of variable-region genes.
Tonegawa’s Bombshell—Immunoglobulin Genes Rearrangement: In 1976, S. Tonegawa and N. Hozumi found the first direct evidence that separate genes encode the V and C regions of immunoglobulins and that the genes are rearranged in the course of B-cell differentiation. This work changed the field of immunology. In 1987, Tonegawa was awarded the Nobel Prize for this work. Selecting DNA from embryonic cells and adult myeloma cells—cells at widely different stages of development— Tonegawa and Hozumi used various restriction endonucleases to generate DNA fragments. The fragments were then separated by size and analyzed for their ability to hybridize with a radio labelled mRNA probe. Two separate restriction fragments from the embryonic DNA hybridized with the mRNA, whereas only a single restriction fragment of the adult myeloma DNA hybridized with the same probe. Tonegawa and Hozumi suggested that, during differentiation of lymphocytes from the embryonic state to the fully differentiated plasma-cell stage (represented in their system by the myeloma cells), the V and C genes undergo rearrangement. In the embryo, the V and C genes are separated by a large DNA segment that contains a restriction-endonuclease site; during differentiation, the V and C genes are brought closer together and the intervening DNA sequence is eliminated.
Multigene Organization of Ig Genes
The К and λ light-chain families contain V, J, and C gene segments; the rearranged VJ segments encode the variable region of the light chains. The heavy-chain family contains V, D, J, and C gene segments; the rearranged VDJ gene segments encode the variable region of the heavy chain. In each gene family, C gene segments encode the constant regions. Each V gene segment is preceded at its 5’ end by a small exon that encodes a short signal or leader (L) peptide that guides the heavy or light chain through the endoplasmic reticulum.
λ-CHAIN MULTIGENE FAMILY: The first evidence that the light-chain variable region was actually encoded by two gene segments appeared when Tonegawa cloned the germ-line DNA that encodes the variable region of mouse λ light chain and determined its complete nucleotide sequence. When the nucleotide sequence was compared with the known amino acid sequence of the λ -chain variable region, an unusual discrepancy was observed. Although the first 97 amino acids of the λ -chain variable region corresponded to the nucleotide codon sequence, the remaining 13 carboxyl-terminal amino acids of the protein’s variable region did not. It turned out that many base pairs away a separate,39-bp gene segment, called J for joining, encoded the remaining 13 amino acids of the λ -chain variable region. Thus, a functional λ variable-region gene contains two coding segments—a 5’ V segment and a 3’ J segment— which are separated by a noncoding DNA sequence in unrearranged germ-line DNA. The λ multigene family in the mouse germ line contains three V λ gene segments, four J λ gene segments, four C λ gene segments.
К-CHAIN MULTIGENE FAMILY: The к-chain multigene family in the mouse contains approximately 85 Vк gene segments, each with an adjacent leader sequence a short distance upstream (i.e., on the 5’ side). There are five Jк gene segments (one of which is a non-functional pseudogene) and a single Cк gene segment. As in the к multigene family, the Vк and Jк gene segments encode the variable region of the к light chain, and the Cк gene segment encodes the constant region. Since there is only one Cк gene segment, there are no subtypes of к light chains.
HEAVY-CHAIN MULTIGENE FAMILY: The organization of the immunoglobulin heavy-chain genes is similar to, but more complex than, that of the К and λ light-chain genes. An additional gene segment encodes part of the heavy-chain variable region. The existence of this gene segment was first proposed by Leroy Hood and his colleagues, who compared the heavy-chain variable-region amino acid sequence with the VH and JH nucleotide sequences.
Variable-Region Gene Rearrangements
The process of variable-region gene rearrangement produces mature, immunocompetent B cells; each such cell is committed to produce antibody with a binding site encoded by the particular sequence of its rearranged V genes. Rearrangements of the heavy chain constant-region genes will generate further changes in the immunoglobulin class (isotype) expressed by a B cell, but those changes will not affect the cell’s antigenic specificity. The steps in variable-region gene rearrangement occur in an ordered sequence, but they are random events that result in the random determination of B-cell specificity.
Light-Chain DNA Undergoes V-J Rearrangements: Expression of both К and λ light chains requires rearrangement of the variable-region V and J gene segments. In humans, any of the functional Vλ genes can combine with any of the four functional Jλ -Cλ combinations. In the mouse, things are slightly more complicated. DNA rearrangement can join the Vλ1 gene segment with either the Jλ1 or the Jλ3 gene segment, or the Vλ2 gene segment can be joined with the Jλ2 gene segment. In human or mouse к light-chain DNA, any one of the Vк gene segments can be joined with any one of the functional Jλ gene segments.
Heavy-Chain DNA Undergoes V-D-J Rearrangements: Generation of a functional immunoglobulin heavy-chain gene requires two separate rearrangement events within the variable region. A DH gene segment first joins to a JH segment; the resulting DHJH segment then moves next to and joins a VH segment to generate a VHDHJH unit that encodes the entire variable region. In heavy-chain DNA, variable-region rearrangement produces a rearranged gene consisting of the following sequences, starting from the 5’ end: a short L exon, an intron, a joined VDJ segment, another intron, and a series of C gene segments. As with the light-chain genes, a promoter sequence is located a short distance upstream from each heavy-chain leader sequence.
Mechanism of Variable-Region DNA Rearrangements - I
Recombination Signal Sequences Direct Recombination: The discovery of two closely related conserved sequences in variable-region germ-line DNA paved the way to fuller understanding of the mechanism of gene rearrangements. DNA sequencing studies revealed the presence of unique recombination signal sequences (RSSs) flanking each germ-line V, D, and J gene segment. One RSS is located 3’ to each V gene segment,5’ to each J gene segment, and on both sides of each D gene segment. These sequences function as signals for the recombination process that rearranges the genes. Each RSS contains a conserved palindromic heptamer and a conserved AT-rich nonamer sequence separated by an intervening sequence of 12 or 23 base pairs. The intervening 12- and 23-bp sequences correspond, respectively, to one and two turns of the DNA helix; for this reason, the sequences are called one-turn recombination signal sequences and two turn signal sequences.
Gene Segments Are Joined by Recombinases: V-(D)-J recombination, which takes place at the junctions between RSSs and coding sequences, is catalyzed by enzymes collectively called V(D)J recombinase. The recombination of variable-region gene segments consists of the following steps, catalyzed by a system of recombinase enzymes:
- Recognition of recombination signal sequences (RSSs) by recombinase enzymes, followed by synapsis in which two signal sequences and the adjacent coding sequences (gene segments) are brought into proximity.
- Cleavage of one strand of DNA by RAG-1 and RAG-2 at the junctures of the signal sequences and coding sequences.
- A reaction catalyzed by RAG-1 and RAG-2 in which the free 3’-OH group on the cut DNA strand attacks the phosphodiester bond linking the opposite strand to the signal sequence, simultaneously producing a hairpin structure at the cut end of the coding sequence and a flush,5’ phosphorylated, double-strand break at the signal sequence.
- Cutting of the hairpin to generate sites for the addition of P-region nucleotides, followed by the trimming of a few nucleotides from the coding sequence by a single strand endonuclease.
- Addition of up to 15 nucleotides, called N-region nucleotides, at the cut ends of the V, D, and J coding sequences of the heavy chain by the enzyme terminal deoxynucleotidyl transferase.
- Repair and ligation to join the coding sequences and to join the signal sequences, catalyzed by normal double strand break repair (DSBR) enzymes.
Mechanism of Variable-Region DNA Rearrangements – II
Productive or Non-productive Gene Rearrangements: One of the striking features of gene-segment recombination is the diversity of the coding joints that are formed between any two gene segments. Although the double-strand DNA breaks that initiate V-(D)-J rearrangements are introduced precisely at the junctions of signal sequences and coding sequences, the subsequent joining of the coding sequences is imprecise. Junctional diversity at the V-J and V-D-J coding joints is generated by a number of mechanisms: variation in cutting of the hairpin to generate P-nucleotides, variation in trimming of the coding sequences, variation in N-nucleotide addition, and flexibility in joining the coding sequences. The introduction of randomness in the joining process helps generate antibody diversity by contributing to the hypervariability of the antigen-binding site. Another consequence of imprecise joining is that gene segments may be joined out of phase, so that the triplet reading frame for translation is not preserved. In such a non-productive rearrangement, the resulting VJ or VDJ unit is likely to contain numerous stop codons, which interrupt translation. When gene segments are joined in phase, the reading frame is maintained. In such a productive rearrangement, the resulting VJ or VDJ unit can be translated in its entirety, yielding a complete antibody. Allelic Exclusion - Single Antigenic Specificity: B cells, like all somatic cells, are diploid and contain both maternal and paternal chromosomes. Even though a B cell is diploid, it expresses the rearranged heavy-chain genes from only one chromosome and the rearranged light-chain genes from only one chromosome. The process by which this is accomplished, called allelic exclusion, ensures that functional B cells never contain more than one VHDHJH and one VLJL unit. This is, of course, essential for the antigenic specificity of the B cell, because the expression of both alleles would render the B cell multi specific. The phenomenon of allelic exclusion suggests that once a productive VHDHJH rearrangement and a productive VLJL rearrangement have occurred, the recombination machinery is turned off, so that the heavy- and light-chain genes on the homologous chromosomes are not expressed.