Do Minigenes Code for All Framework Segments of Immunoglobulin Chains

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-

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“…6; C. A. Reynaud and J. C. Weill, personal communication), resembling the minigene hypothesis initially suggested by Kabat et al. (7).…”

supporting

confidence: 75%

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

Hsu

Schwager

Alt

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…It allows, for example, as exemplified by the present variant, to re-assort CDRs within similar frameworks. Kabat and his coworkers (Kabat et al, 1978(Kabat et al, , 1979 have summarized the evidence for such reassortment as derived from V region amino acid sequences. It also assigns a biological role to "pseudo" V genes which appear to represent a large fraction of the V genes carried in the germ line (Givol et al, 1981;Bothwell et al, 1981).…”

Section: Isolation Of Cyanogen Bromidefragmentsmentioning

confidence: 99%

Immunoglobulin V region variants in hybridoma cells. II. Recombination between V genes.

Dildrop¹,

Brüggemann²,

Radbruch³

et al. 1982

The EMBO Journal

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The mouse hybridoma line B1-8.61 secretes a monoclonal IgD, Xl anti44-hydroxy-3-nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V-region variants (B1-8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1-8.61 and variant heavy chain V regions were deter-

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“…Moreover, another cloned VH region (S107) hybridizes with a different set of VH genes, which exhibits very little if any overlap with the VH76 set (our unpublished results). If each of the 5-10 subgroups already evident from amino acid sequences of VH regions (10,12) are encoded by a comparable number of genes, then there must be a minimum of 40-80 germline VH genes. Thus, there is now direct evidence at the nucleic acid level that multiple related VH genes in the germline contribute to immunoglobulin diversity.…”

Section: Resultsmentioning

confidence: 99%

“…The three independent families of immunoglobulin genes include one each for the K and X light chains and one for all types of heavy chains, such as ji, a, and y. Indirect evidence that the mouse genome contains multiple V, and VH genes has come from attempts to place known amino acid sequences (10) into "subgroups" on the basis of sequence homology. Because the sequences in different subgroups differ substantially, each subgroup presumably requires at least one germline V gene (11,12).…”

mentioning

confidence: 99%

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

Kemp¹,

Cory²,

Adams³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Immunoglobulins are composed of light and heavy (H) chains, each of which contains a variable (V) and a constant (C) region. The longstanding hypothesis (1) that V and C regions are encoded by separate genes in the germline has recently been confirmed by molecular cloning (2-9). The three independent families of immunoglobulin genes include one each for the K and X light chains and one for all types of heavy chains, such as ji, a, and y. Indirect evidence that the mouse genome contains multiple V, and VH genes has come from attempts to place known amino acid sequences (10) into "subgroups" on the basis of sequence homology. Because the sequences in different subgroups differ substantially, each subgroup presumably requires at least one germline V gene (11,12). Direct evidence for multiple VK genes in the mouse genome has recently been obtained by cloning (5-7), but hitherto such evidence has not been available for the VH genes. Classical genetic approaches suggest that the VH genes are linked both to each other and to CH genes (13), but such studies cannot establish the physical distance between genes nor provide any detailed picture of V gene organization. Molecular analysis of V gene linkage might demonstrate whether they are arranged as regular repeating units, as are the members of some other multigene families (12), and might reveal features of their organization relevant to the generation of immunoglobulin diversity.Hitherto, individual immunoglobulin genes have been cloned in phage X vectors by insertion of mouse DNA fragments generated by digestion with EcoRI (2-9). This approach has the limitation that any particular gene may occur in an EcoRI fragment too small to span a region of interest or too large to fit into a phage vector. This limitation can be overcome by cloning a set of large overlapping sequences. Maniatis et al. (14) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4627 have used this approach to construct "clone libraries" extensive enough to encompass an entire mammalian genome by attaching randomly fragmented DNA to the phage X vector Charon 4A (15) with synthetic linkers. Because the clones in such a library have overlapping sequences, they are particularly appropriate to studies of gene linkage. We report here the construction of a library of cloned segments of the mouse genome by an approach that obviates the need for linkers. As a start towards examining the organization of immunoglobulin heavy chain genes, we have searched this library with a cloned cDNA copy of a ,u mRNA. At least eight distinct VH genes are represented in the clones examined so far, demonstrating that multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of two clones bears a pair of VH genes, separated by a spacer DNA sequence some 40 times the length of the VH genes. This indicates that related VH gen...

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Do Minigenes Code for All Framework Segments of Immunoglobulin Chains

Cited by 32 publications

References 10 publications

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

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

Immunoglobulin V region variants in hybridoma cells. II. Recombination between V genes.

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

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