BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 33. IMMUNOGLOBULINS

33.18. Variable and Constant Regions Are Encoded by Separate But Joined Genes

How are immunoglobulin genes organized and how do they function? The discovery of a clear distinction between variable and constant regions in L and H chains suggested that immunoglobulin genes, much like the Polypeptides they encode, have an unusual architecture. As mentioned previously, the constant regions of x chains are identical at all positions except 191, which can be occupied by either leucine or valine. Pedigree analysis has shown that these two variants (allotypes) are inherited according to classical Mendelian rules, providing strong evidence that the constant region of x chains is encoded by a single Gene. In contrast, genetic analyses indicate that variable regions are encoded by multiple genes. In 1965, William Dreyer and Claude Bennett proposed that in germ-line Cells, multiple V genes are spatially separated from a single C gene. According to this hypothesis, during the differentiation of an antibody-producing Cell, one of the V genes joins with the C gene. It was later discovered that DNA splicing can indeed occur, analogous to the incorporation of phage DNA into the host genome, for example.

Definitive testing of this translocation hypothesis had to await the ISOLATION OF PURE immunoglobulin mRNA and The Development of Methods for analyzing complex mammalian genomes. Of particular interest in this regard was The Use of restriction Enzymes, which cleave large Chromosomes into specific DNA fragments that can be readily analyzed further. The distribution of V and C genes within these DNA fragments can be determined by hybridizing them with mRNA fragments specific for either the variable or constant region. In 1976, Susumu Tonegawa used this approach to demonstrate that in germ-line DNA, the V and C genes are located far apart, whereas in antibody-producing cells, they are closely linked. Consequently, during lymphocyte differentiation, immunoglobulin genes undergo rearrangement (Fig. 33.23).

Class="center">Fig. 33.23. During the differentiation of antibody-producing cells, the V gene translocates and joins with the C gene

33.19. How Does Antibody Specificity Diversity Arise?

An animal's Organism is capable of synthesizing large amounts of specific Antibodies within a few weeks after the administration of virtually any foreign determinant. The number of different antibody types that an animal can synthesize is enormous—likely exceeding one million. As we have seen, the structural basis for antibody Specificity resides in the Amino acid sequences of the variable regions of light and heavy chains. This brings us to a key question: how do the diverse amino acid sequences of the variable regions arise? More specific questions can also be formulated in this regard.

1. When does diversity arise? During the lifetime of the animal (somatic) or over the course of evolution (genetic)?

2. How does diversity arise? Through random Mutations during evolution, via somatic recombination, or through somatic hypermutation?

A wealth of data regarding The amino acid sequences of myeloma IMMUNOGLOBULINS has allowed immunologists to formulate several hypotheses concerning How Antibody Diversity originated. Three possible mechanisms have been proposed.

1. The germ-line theory. According to this model, diversity is already inherent in germ-line cells (and embryonic cells), which contain

a very large number (>10⁴) of genes encoding antibody variable regions. Each unique variable region sequence corresponds to a DNA segment in the germ-line cells. Thus, this hypothesis suggests that diversity arose during evolution through random mutation and Selection.

2. The somatic recombination theory. According to this hypothesis, there is only a small number (on the order of 100) of genes encoding antibody variable regions. These genes, which are similar but not identical, undergo repeated recombination events in antibody-producing cells during the individual's lifetime. Crossing-over is postulated to occur intrachromosomally. Calculations have shown that the recombination of 10 genes can lead to the generation of 10⁶ different amino acid sequences.

3. The somatic hypermutation theory. According to this model, diversity arises As a result of point mutations in a single variable region gene of a given subclass. However, only an unusually high mutation rate could generate the full DIVERSITY OF ANTIBODY specificity within an individual's lifetime. It has been suggested that in antibody-producing cells (or their precursors), the elevated mutation rate is maintained by a DNA Repair mechanism that is error-prone in this context.

33.20. Variable Regions of L and H Chains Are Encoded by Several Hundred Genes

The revolution in DNA study methods over recent years has made THE ORIGIN OF antibody diversity amenable to experimental analysis. Gene cloning techniques and rapid methods for sequencing large DNA segments have proven particularly valuable. Through the use of these techniques, information required to answer two pivotal questions was rapidly obtained: how many variable region genes are present in germ-line cells? And does The base sequence change during the differentiation of antibody-producing cells?

The answer to the first question is that there are several hundred variable region genes for x light (Vx) and heavy (VH) chains. The 300 Vx genes and 300 V_H genes, by combining in various ways (300 × 300), could theoretically encode 9 • 10⁴ antibody variants with different specificities. This calculated maximum (9 • 10⁴) is far lower than the actual number of antibodies of various specificities synthesized in an animal organism; it is estimated that the number of such antibodies significantly exceeds 10⁶. The discrepancy between the theoretical calculation and the actual value is even greater for light chains, which have been shown to be encoded by fewer than 10 V, genes. Overall, the number of variable region genes in germ-line cells proved too small to fully account for the totality of antibody diversity. Evidently, additional factors that enhance the degree of diversity arise during lymphocyte differentiation over the lifespan of the animal.

33.21. The Discovery of J (Joining) Genes—An Additional Source of Antibody Diversity

The next step in elucidating the mechanism responsible for antibody diversity was made by determining the base sequences of cloned genes encoding immunoglobulins in embryonic and myeloma cells. These exceptionally fruitful studies were conducted by Tonegawa, Philip Leder, and Leroy Hood. First, quite unexpectedly, it turned out that in embryonic cells, V genes do not encode the entire L and H chain variable regions. The V gene of embryonic cells (and germ-line cells) ends with the code for amino acid residue 95, rather than residue 108, which marks the end of the immunoglobulin polypeptide chain's variable region (Fig. 33.24). Where is the DNA that encodes the final 13 residues of the variable region located? In embryonic cells, this DNA segment is situated in an unexpected Location: close to the C gene. This DNA segment was named the J gene (for joining) because in differentiated cells it joins the V and C genes. In essence, embryonic cells contain an entire cluster of tandemly arranged J genes near the C gene (Fig. 33.25). During the differentiation of antibody-producing cells, the V gene translocates to a region adjacent to the C gene; this translocation is accomplished via intrachromosomal recombination. In the process, the V gene is spliced to the J gene, forming a composite gene that fully encodes the variable domain. Each of these genes contains a short palindromic sequence (Section 24.27) located adjacent to the recombination site. These palindromes likely serve as recognition elements during the recombination process.

Fig. 33.24. The V gene isolated from embryonic cells is truncated. It does not encode the final 13 amino acid residues of the variable (V) region of the polypeptide chain

Fig. 33.25. Tandem arrangement of a group of J genes encoding a portion of the last hypervariable region of the variable domain; the J genes are located near the C gene

J genes make a major contribution to antibody diversity by encoding part of the final hypervariable region of both L and H chains. During the assembly of a complete V_ӽ gene, any of several hundred V genes can join to any of the five J genes. For example, the combination of 300 incomplete V genes with five J genes can yield 1,500 Variants of the complete (continuous) V gene. Consequently, somatic recombination of these gene segments further enhances the diversity already established in germline cells.