Evolution of immunoglobulin genes: VH families in the amphibian Xenopus.

Abstract

We have isolated multiple independent cDNA clones that represent mRNA sequences of immunoglobulin heavy chains from the spleen of adult Xenopus laevis. These cDNA clones contained constant (C) region sequences that were either C,, or a separate C region sequence believed to be C,, In individual cDNA clones the C region sequences were associated with independent heavy-chain variable region (VH) sequences that were classifiable into five distinct families. Genomic Southern blotting analyses with family-specific probes indicated that the haploid genome contains a minimum of 80 VH gene segments, a number similar to that found in various mammalian species. Multiple JH and putative DH segments were also identified (J, joining; D, diversity). Analyses of 13 independent VHDJH junctions suggest that combinatorial and junctional diversification mechanisms probably arose early in vertebrate evolution. Finally, comparison of Xenopus VH sequences to those from other vertebrates indicated conservation of V region framework residues that are responsible for the tertiary structure of the Fv throughout evolution.The basic four-chain unit of an immunoglobulin molecule is composed of two heavy (H) and two light (L) chains, the N-terminal (variable, V) regions of which interact to form the antigen-binding site. The immunoglobulin H-chain V region gene (VH) is formed by the joining of three gene segments-VH, DH, and JH-while the L-chain V region gene (VL) is assembled from VL and JL segments (D, diversity; J, joining). These rearranged units are assembled upstream from exons that encode the respective H-or L-chain constant (C) regions and are then transcribed to generate the H-or L-chain mRNA. The antigen contact regions of the H-and L-chain V regions are referred to as complementarity-determining regions (CDRs), which were originally defined from variability plots of numerous V region amino acid sequences (1, 2). Each V region has three CDRs, two of which are encoded in the germ-line V segment (CDR1 and CDR2); the other (CDR3) is encoded by the VDJ and VJ junctional regions.Mammals, chickens, and sharks all assemble and express immunoglobulin H-and L-chain genes by the basic processes outlined above but they differ radically in gene organization and mechanisms to generate an extraordinary diversity of specificities in antibody molecules. The mouse and human generate diversity through a combination of germ-line and somatic processes in which large numbers of germ-line gene segments arranged in tandem as multiple V (containing CDR1 and CDR2), multiple D, and multiple J segments are rearranged somatically in different combinations (to create different CDR3s); in addition, diversity is further augmented by somatic processes of junctional diversification and somatic hypermutation (3, 4). The shark also contains 100-200 germ-

What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

“…The organization of Ig genes in Xenopus closely resembles that of mammals, and there is no shortage of the elements needed for combinatorial diversity (Hsu et al, 1989;Schwager et al, 1989Schwager et al, , 1991aHaire et al, 1990). Moreover, as we confirm here (Figure 3), there is no shortage of junctional diversity (Schwager et al, 1991a (Wabl et al, 1985;Berek and Milstein, 1988;Kocks and Rajewsky, 1989).…”

“…The Xenopus D cluster has yet to be mapped and, so far, D15 is the only germline D segment, TGGTAC, to be sequenced (clone G2, Schwager et al, 1991a). However, D sequences have been deduced from a large number of cDNA clones (Schwager et al, 1988a,b;Hsu et al, 1989;Haire et al, 1990) …”

“…Genes encoding both H and L chains have been sequenced, and there is no lack of potential elements for rearrangement (Schwager et al, , 1991aHsu et al, 1989;Haire et al, 1990;Zezza et al, 1991). As in mammals (reviewed by Tonegawa, 1983), the third complementarity determining region (CDR3) is the most diverse.…”

IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates

“…Because of this large predicted size and relatively low expression, a complete ␦ cDNA was not isolated from our libraries, and no EST was found in the public databases. Thus, as described in Materials and Methods, we used PCR to amplify clones from X. tropicalis spleen RNA using primers specific for C8 exon, TM, and 3Ј-UTR, as well as primers predicted to amplify the majority of Xenopus V gene families (23). Most cDNAs were found to encode nine-domain-long molecules containing TM regions, expected from the genomic scaffold (all coding regions shown in Fig.…”

Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis.

“…The extended length of some D regions (e.g., 26035 and 26510) suggests that two D segments may contribute to a single rearrangement event as can occur in Heterodontus (9) (Hinds, K., and G. Litman, unpublished data) and possibly in an avian (54). Joining of two D segments also may occur in XL (37) . In addition, 13 D segments are unassigned with regard to the primary families.…”