Structure and multiplicity of genes for the human immunoglobulin heavy chain variable region.

Abstract

Several genes for the variable region of immunoglobulin heavy chains (VH genes) have been isolated from human fetal liver DNA by using a cDNA plasmid probe containing a mouse VH sequence. The detectable VH genes are separated by [12][13][14][15][16] kilobases of DNA, and hybridization experiments show about 23 hybridizing VH genes in DNA of three different individuals. The complete nucleotide sequence of one of these human VH genes shows that it belongs to the human VHIII subgroup. The VH gene appears to contain an intervening sequence (104 bases in length) within a precursor sequence, between residues -4 and'-5. The precursor sequence is itself 19 codons in length. The 3' end ofthe V gene seems to be at codon 93 or 94, and this is followed by the conserved sequences C-A-C-A-G-T-G and G-A-CA--A-A-A-C-C. The presence of these sequences suggests that similar enzymatic mechanisms are involved in the integration of V genes in both heavy and light chains. Immunoglobulins are made of two types of polypeptide chain, the heavy (H) and the light (L) chains, and each of these chains has an NH2-terminal variable (V) region covalently attached to a COOH-terminal constant (C) region. The variability in the sequence of the V regions of antibodies is the feature that gives rise to the diversity of the antibody response, and an outstanding problem is the extent of inherited V gene diversity compared to additional diversity that may be generated by somatic means. In addition, as predicted by the two-genes-one-polypeptide hypothesis (1), the V genes are inherited as a separate set of genes that are linked to the respective C genes (2). In mouse K light chains it is clear that a set of VK genes exists as a separate unit from a CK gene; integration of one of the VK genes and the CK gene results in the functional K chain gene (3)(4)(5). Nucleotide sequence data of VX (6) and VK (7-9) L chains has shown that the V gene sequence terminates at the codon for amino acid 96. The rest of the V region gene comes from one of a set of joining segments (JL). After VL-JL integration the fused V gene remains separated from the C gene by a large intervening sequence (4, 7-9). A further intervening sequence occurs within the precursor region of both VA and VK genes, and all these sequences are transcribed and subsequently removed from the nuclear RNA (10,11 MATERIALS AND METHODSThe human fetal liver phage library was kindly given by T. Maniatis and was constructed by Hae III/AIu I digestion of DNA followed by the addition of EcoRI linker molecules and insertion into X Charon 4A (12). Plaque hybridization (13) of the library was carried out after plating on Nunc bioassay plates (23 X 23 cm), using the nick-translated plasmid pg/107 (14).Restriction endonuclease mapping of purified positive phage was carried out by using single and double digests of EcoRI, Kpn I, Bgl II, Bst I, and Xho I, comparing sizes to those of the X Charon 4A host (15), hybridizing, and determining the nucleotide sequence. Southern filter hybridization analysis (16) ...

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