Divergent Evolution and Evolution by the Birth-and-Death Process in the Immunoglobulin VH Gene Family Tatsuya Ota and Masatoshi Nei Department of Biology and Institute of Molecular Evolutionary Genetics, The Pennsylvania State University Immunoglobulin diversity is generated primarily by the heavy- and light-chain variable-region gene families. To understand the pattern of long-term evolution of the heavy-chain variable-region (V,) gene family, which is composed of a large number of member genes, the evolutionary relationships of representative Vn genes from diverse organisms of vertebrates were studied by constructing a phylogenetic tree. This tree indicates that the vertebrate Vn genes can be classified into group A, B, C, D, and E genes. All Vn genes from cartilaginous fishes such as sharks and skates form a monophyletic group and belong to group E, whereas group D consists of bony- fish Vn genes. By contrast, group C includes not only some fish genes but also amphibian, reptile, bird, and mammalian genes. Group A and B genes were composed of the genes from mammals and amphibians. The phylogenetic analysis also suggests that mammalian Vu genes are classified into three clusters-i.e., mammalian clans I, II, and III-and that these clans have coexisted in the genome for >400 Myr. To study the short-term evolution of Vn genes, the phylogenetic analysis of human group A (clan I) and C (clan III) genes was also conducted. The results obtained show that Vn pseudogenes have evolved much faster than functional genes and that they have branched off from various functional Vu genes. There is little indication that the Vu gene family has been subject to concerted evolution that homogenizes member genes. These observations indicate that the Vn genes are subject to divergent evolution due to diversifying selection and evolution by the birth-and-death process caused by gene duplication and dysfunctioning mutation. Thus, the evolutionary pattern of this monofunctional multigene family is quite different from that of such gene families as the ribosomal RNA and histone gene families. Introduction Monofunctional multigene families, where all Concerted evolution was also invoked to explain member genes have the same function, are generally be- the evolution of the immunoglobulin heavy-chain vari- lieved to undergo coincidental or concerted evolution, able-region (Vu) genes (Hood et al. 1975; Ohta 1980, which homogenizes the DNA sequences of the member 1983). Gojobori and Nei ( 1984), however, showed that genes (Smith et al. 197 1; Smith 1974; Zimmer et al. Vu gene (often called “gene segment”) family in the 1980). Good examples of concerted evolution are seen mouse and human undergoes a very slow rate of con- with ribosomal RNA (rRNA) genes (e.g., see Arnheim certed evolution, if any; that the extent of sequence di- 1983) and histone genes (e.g., see Matsuo and Yamazaki vergence between member genes in the mouse or the 1989)) where all the member genes have virtually iden- human is substantial; and that some of these genes di- tical coding sequences within species. This is under- verged much earlier than the mammalian radiation standable, because these gene families produce a large ( N 80 Mya) (also see Tutter and Riblet 1989a). quantity of gene products of the same function that are During the past 10 years, the DNA sequences of essential for the survival of an organism. Concerted evo- Vu genes have been determined for many lower verte- lution is caused either by unequal crossover or intergenic brate organisms (see Litman et al. 1993 ) . We can there- fore study the pattern of evolution of the Vu gene family gene conversion. on a much longer evolutionary time scale. This type of study is particularly interesting, because the gene orga- Key words: birth-and-death process, divergent evolution, im- nization of Vu genes is now known to vary extensively munoglobulin, multigene family, VH genes. among different classes of vertebrates (Du Pasquier Address for correspondence and reprints: Masatoshi Nei, Institute 1993). In this paper, we show that the pattern of Vu of Molecular Evolutionary Genetics, The Pennsylvania State University, gene evolution is very different from the typical con- 328 Mueller Laboratory, University Park, Pennsylvania 16802. certed evolution and that two evolutionary processes, Mol. Biol. Evol. 11(3):469-482. 1994. 0 1994 by The University of Chicago. All rights reserved. which may be called “divergent evolution” and “evo- 0737-4038/94/l 103~0014$02.00 lution by the birth-and-death process,” predominate in 469 470 Ota and Nei Ancestral form VDJ C Cartilaginous fishes Higher vertebrates I IV -IHHHHW .: ,: B V DDJ C VDDJ C VDDJ C Vn Dn Jn Cn Horned shark Mammals V DDJ C VDDJ C VDDJ C vVn V Dn J Cn Little skate Chicken FIG.1.-Genomic organizations of immunoglobulin heavy-chain genes. Cartilaginous fishes have the ( VH-DH-Dn-JH-CH)ntype organization including germ-line-joined genes, whereas most higher vertebrates, including bony fishes and tetrapods, have the (Vn),-( Dn)“-( Jr_,)“-(Cn), type gene organization. Chicken has a single functional variable gene. V = variable-segment gene; D = diversity-segment gene; J = joining-segment gene; C = constant-segment gene; and w = pseudogene. For more detailed information, see the work of Litman et al. ( 1993). the evolution of Vn genes in vertebrates, despite the fact The VH (as well as V,) genes can be subdivided that these genes have the same function. We first study further into two hypervariable or complementarity-de- the long-term evolution of VH genes, examining the termining regions (CDR 1 and CDR2 ) and three frame- evolutionary relationships of the genes obtained from work regions (FWl, FW2, and FW3). (CDR3 is deter- various classes of vertebrates, and then a short-term mined primarily by the Dn and Jn genes, so it was evolution of human Vn genes. excluded in this paper). The human and mouse genomes contain > 100 VH genes and a few to a dozen Dn and Material and Methods Jn genes (see fig. 1). Each of the Vn’s is recombined Background Information with one of the Du’s, one of the Jn’s, and one of the Immunoglobulin or antibody molecules consist of Cn’s (constant region genes), to form a heavy-chain gene two heavy chains and two light chains. Both the heavy (Tonegawa 1983). This process therefore generates a and light chains are composed of the variable region and large number of different immunoglobulins that are the constant region (C). The variable region is respon- necessary for defending an individual from many dif- sible for antigen-binding, whereas the constant region is ferent foreign antigens. Essentially the same humoral responsible for effector function. The heavy-chain vari- immune system exists for most of the bony fishes and able region is encoded by the variable-segment ( Vn ) , tetrapods. diversity-segment (Dn), and joining-segment (Jn) genes, In lower vertebrates such as sharks and skates, whereas the light-chain variable region is encoded by the however, the genomic organization of the immunoglob- variable-segment (V,) and joining-segment ( JL) genes. ulin genes is different from that of higher vertebrates, However, the major portion of the variable region is which may be described as (V,-D,-J,-C,) . In lower ver- determined by either Vn or VL. The Vn and Vi_ genes tebrates, the basic unit of repeat of genes is (V-D-D-J- are evolutionarily homologous with each other, but their C) (fig. 1); that is, the linked gene group Vn-Dn-Dn-Ju- sequence similarity is quite low. Cu is repeated several hundred times in the genome, Evolution of Immunoglobulin VH Gene Family 47 1 and the repeat seems to be scattered on different chro- from each species and to cover a wide range of organisms. mosomes (Litman et al. 1993). Therefore, the immu- We have therefore included (a) almost all sequences noglobulin genes in these organisms may be represented from a species where a small number of genes have been by (V-D-D-J-C),, . studied but (b) a relatively few representative sequences Of course, there are many exceptions to this rule. from a species where a large number of genes have been Some units of shark genes have the Vu and Du genes studied. For the study of long-term evolution, no pseu- that are already united (VDD-J-C) in the germ-line ge- dogenes were included. In both human and mouse, >60 nome, whereas in some other units all VH , Du’s, and JH functional germ-line genes have been sequenced, but are united (VDDJ-C) (Litman et al. 1993). In the little Schroeder et al. ( 1990) showed that they can be classified skate (Raja erinacea), (VD-DJ-C) are also present. into three major clusters, i.e., clans I-III (also see Kirk- Furthermore, in the coelacanth (Latimeria chalumnae), ham et al. 1992). Our preliminary analysis of -600 which belongs to the lobe-finned fishes, (V-D) is appar- mammalian VH genes, which were obtained from ently repeated many times in the 5’ side of the J and C GenBank and Kabatnuc databases, has also suggested genes ( Amemiya et al. 1993). Actually, the number of that these genes can be grouped into the three major lower vertebrates studied so far is very small, so it is clusters (data not shown). We have therefore used 11 likely that some new types of genomic organization of and 7 representative sequences from the mouse and the Vu genes will be discovered in the near future. human, respectively, covering the three major clusters, Another group of vertebrates that has a deviant ge- i.e., clans I-III. More than 30 different Vn sequences nomic organization of the VH genes is that of avian spe- are also available from the African toad Xenopus laevis.