8536d_ch05_105-136 8/1/02 8: 53 AM Page 107 mac79 Mac 79:45_Bw Glasby et al. Immunology 5e Organization and Expression of Immunoglobulin Genes CHAPTER 5 107 immunoglobulin genes had to account for the following in rabbits by C. Todd, who found that a particular allotypic properties of antibodies marker in the heavy-chain variable region could be associ ated with a, Y, and u heavy-chain constant regions. Consid- The vast diversity of antibody specificities rable additional evidence has confirmed that a single The presence in Ig heavy and light chains of a variable ariable-region sequence, defining a particular antigenic region at the amino-terminal end and a constant region specificity, can be associated with multiple heavy-chain at the carboxyl-terminal end constant-region sequences; in other words, different classes, or isotypes, of antibody (e.g. IgG, IgM)can be expressed a The existence of isotypes with the same antigenic with identical variable-region sequences specificity, which result from the association of a given variable region with different heavy-chain constant region Dreyer and Bennett Proposed the Two-Gene Model Germ-Line and Somatic- Variation Models In an attempt to develop a genetic model consistent with the Contended To Explain Antibody Diversity known findings about the structure of immunoglobulins, w Dreyer and J. Bennett suggested, in their classic theoretical For several decades, immunologists sought to imagine a ge- paper of 1965, that two separate genes encode a single netic mechanism that could explain the tremendous diversity munoglobulin heavy or light chain, one gene for the V region of antibody structure. Two different sets of theories emerged. (variable region) and the other for the C region(constant re The germ-line theories maintained that the genome con- gion). They suggested that these two genes must somehow tributed by the germ cells, egg and sperm, contains a large come together at the dna level to form a continuous mes repertoire of immunoglobulin genes; thus, these theories in- sage that can be transcribed and translated into a single Ig voked no special genetic mechanisms to account for anti- heavy or light chain. Moreover, they proposed that hundreds body diversity. They argued that the immense survival value or thousands of V-region genes were carried in the germ line, of the immune system justified the dedication of a significant whereas only single copies of C-region class and subclass fraction of the genome to the coding of antibodies In con- genes need exist trast,the somatic-variation theories maintained that the The strength of this type of recombinational model genome contains a relatively small number of immunoglob- (which combined elements of the germ-line and somatic- ulin genes, from which a large number of antibody specifici- variation theories) was that it could account for those im ties are generated in the somatic cells by mutation or munoglobulins in which a single V region was combined recombination with various C regions. By postulating a single constant As the amino acid sequences of more and more im- region gene for each immunoglobulin class and subclass, the munoglobulins were determined, it became clear that there model also could account for the conservation of necessary sity but also for maintaining constancy. Whether diversity diversification of variable-region genes g for evolutionary must be mechanisms not only for generating antibody diver- biological effector functions while allowi was generated by germ-line or by somatic mechanisms, a At first, support for the Dreyer and Bennett hypothesis paradox remained: How could stability be maintained in the was indirect. Early studies of DNA hybridization kinetics us- constant(C)region while some kind of diversifying mecha- ing a radioactive constant-region DNA probe indicated that nism generated the variable(V)region? the probe hybridized with only one or two genes, confirming Neither the germ-line nor the somatic-variation propo- the models prediction that only one or two copies of each nents could offer a reasonable explanation for this central constant-region class and subclass gene existed. However, in- feature of immunoglobulin structure Germ-line proponents direct evidence was not enough to overcome stubborn resis- found it difficult to account for an evolutionary mechanism tance in the scientific community to the hypothesis of Dreyer that could generate diversity in the variable part of the many and Bennet. The suggestion that two genes encoded a single heavy-and light-chain genes while preserving the constant polypeptide contradicted the existing one gene-one region of each unchanged. Somatic-variation proponents polypeptide principle and was without precedent in any found it difficult to conceive of a mechanism that could di- known biological system. versify the variable region of a single heavy- or light-chain As so often is the case in science, theoretical and intellec gene in the somatic cells without allowing alteration in the tual understanding of lg-gene organization progressed ahead amino acid sequence encoded by the constant region. of the available methodology. Although the Dreyer and Ben- A third structural feature requiring an explanation nett model provided a theoretical framework for reconciling emerged when amino acid sequencing of the human the dilemma between Ig-sequence data and gene organiza myeloma protein called Til revealed that identical variable- tion, actual validation of their hypothesis had to wait for sev- region sequences were associated with both y and u heavy- eral major technological advances in the field of molecular chain constant regions. A similar phenomenon was observed biologyimmunoglobulin 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 Contended To Explain Antibody Diversity For several decades, immunologists sought to imagine a ge￾netic mechanism that could explain the tremendous diversity of antibody structure. Two different sets of theories emerged. The germ-line theories maintained that the genome con￾tributed by the germ cells, egg and sperm, contains a large repertoire of immunoglobulin genes; thus, these theories in￾voked no special genetic mechanisms to account for anti￾body 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 con￾trast, the somatic-variation theories maintained that the genome contains a relatively small number of immunoglob￾ulin genes, from which a large number of antibody specifici￾ties are generated in the somatic cells by mutation or recombination. As the amino acid sequences of more and more im￾munoglobulins were determined, it became clear that there must be mechanisms not only for generating antibody diver￾sity but also for maintaining constancy. Whether diversity was generated by germ-line or by somatic mechanisms, a paradox remained: How could stability be maintained in the constant (C) region while some kind of diversifying mecha￾nism generated the variable (V) region? Neither the germ-line nor the somatic-variation propo￾nents 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 di￾versify 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 and heavy￾chain constant regions. A similar phenomenon was observed in rabbits by C. Todd, who found that a particular allotypic marker in the heavy-chain variable region could be associ￾ated with , , and heavy-chain constant regions. Consid￾erable additional evidence has confirmed that a single variable-region sequence, defining a particular antigenic specificity, can be associated with multiple heavy-chain constant-region sequences; in other words, different classes, or isotypes, of antibody (e.g., IgG, IgM) can be expressed with identical variable-region sequences. Dreyer and Bennett Proposed the 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 im￾munoglobulin heavy or light chain, one gene for the V region (variable region) and the other for the C region (constant re￾gion). They suggested that these two genes must somehow come together at the DNA level to form a continuous mes￾sage 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 im￾munoglobulins 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. At first, support for the Dreyer and Bennett hypothesis was indirect. Early studies of DNA hybridization kinetics us￾ing a radioactive constant-region DNA probe indicated that the probe hybridized with only one or two genes, confirming the model’s prediction that only one or two copies of each constant-region class and subclass gene existed. However, in￾direct evidence was not enough to overcome stubborn resis￾tance in the scientific community to the hypothesis of Dreyer and Bennet. The suggestion that two genes encoded a single polypeptide contradicted the existing one gene–one polypeptide principle and was without precedent in any known biological system. As so often is the case in science, theoretical and intellec￾tual understanding of Ig-gene organization progressed ahead of the available methodology. Although the Dreyer and Ben￾nett model provided a theoretical framework for reconciling the dilemma between Ig-sequence data and gene organiza￾tion, actual validation of their hypothesis had to wait for sev￾eral major technological advances in the field of molecular biology. Organization and Expression of Immunoglobulin Genes CHAPTER 5 107 8536d_ch05_105-136 8/1/02 8:53 AM Page 107 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: