Rearrangement of immunoglobulin light chain and heavy chain constant region genes in multiple myeloma]

We analysed immunoglobulin (Ig) gene rearrangements in 28 patients with multiple myeloma by Southern hybridization method. We used 5 probes which cover C kappa and kappa de loci of Ig light chain kappa gene, and JH, 5'S mu and S gamma 3 loci of Ig heavy chain gene. In 11 out of 12 patients with kappa-producing myeloma, DNA rearrangements were observed using C kappa probe. Among them, kappa de region was rearranged in 7 patients and kept germline configuration in 4 patients. In all of 14 patients with lambda-producing myeloma, C kappa region was deleted and kappa de region was rearranged. 5'S mu-probe was very useful for detecting class switch recombination, and furthermore by using S gamma-probe together, S mu-S gamma joining could be detected. In all of 10 patients with gamma-producing myeloma, 5'S mu and S gamma-probes detected the rearranged band of the same size on at least 1 allele, which suggested the presence of S mu-S gamma joinings. In 8 of 10 patients with Bence-Jones myeloma, 5'S mu-probe detected rearranged bands and the presence of class switch recombinations were suggested as observed in other Ig secretory myelomas. In other 2 patients with Bence-Jones myeloma, non-functional class switch recombinations were detected. The results of this study indicated that genotypes corresponded well to phenotypes in multiple myeloma, and further analysis in other types of B cell malignancies will be interesting.     

Amino acid sequence of the precursor region of MOPC-315 mouse immunoglobulin heavy chain.

Partially purified mRNA coding for the MOPC-315 heavy immunoglobulin alpha chain was translated in a reticulocyte lysate containing 20 labeled amino acids. Radiolabeled MOPC-315 heavy chain precursor protein, purified by preparative gel electrophoresis and immunoprecipitation, was sequenced by Edman degradation. The labeled phenylthiohydantoin amino acid obtained in each cycle was identified and quantitated by high-pressure liquid chromatography. The precursor sequence of 18 amino acids, Met-Lys-Val-Leu-Ser-Leu-Leu-Tyr-Leu-Leu-Thr-Ala-Ile-Pro-His-Ile-Met-Ser, preceded the sequence corresponding to the NH2 terminus of the mature secreted heavy chain.     

Linkage and sequence homology of two human immunoglobulin gamma heavy chain constant region genes.

We report the nucleotide sequence of a gene encoding a human immunoglobulin C gamma 2 region. Comparison with the previously determined C gamma 4 sequence reveals that these two genes share extensive (approximately 95%) homology in the three CH domain exons and adjacent noncoding regions. In contrast, hinge exons have diverged to a much greater degree, implying that natural selection has favored the generation of diversity in these coding regions. We have used the noncoding nucleotide differences to estimate that approximately 6-7 million years have elapsed since the occurrence of the gene duplication or correction event which generated the two identical ancestral genes. In addition we show that the two C gamma genes are arranged in human chromosomal DNA in the configuration 5'-C gamma 2-17 kilobase pairs -C gamma 4-3'.     

Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions.

A high-molecular-weight DNA from Balb/c mouse early embryo or from MOPC 321 plasmacytoma (a k-chain producer) was digested to completion with Bacillus amyloliquefaciens strain H restriction enzyme (BamH I). The resulting DNA fragments were fractionated according to size in preparative agarose gel electrophoresis. DNA fragments carrying gene sequences coding for the variable or constant region of k chains were detected by hybridization with purified, 125I-labeled, whole MOPC 321 K MRNA and with its 3'-end half. The pattern of hybridization was completely different in the genomes of embryo cells and of the plasmacytoma. The pattern of embryo DNA showed two components, one of which (molecular weight=6.0 million) hybridized with C-gene sequences and the other (molecular weight=3.9 million) with V-gene sequences. The pattern of the tumor DNA showed a single component that hybridized with both V-gene and C-gene sequences and that is smaller (molecular weight=2.4 million) than either of the components in embryo DNA. The results were interpreted to mean that the Vk and Ck genes, which are some distance away from each other in the embryo cells, are joined to form a contiguous polynucleotide stretch during differentiation of lymphocytes. Such joining occurs in both of the homologous chromosomes. Relevance of these findings with respect to models for V-C gene joining, activation of a specific V k gene, and allelic exclusion in immunoglobulin gene loci is discussed.     

Partial amino-acid sequence of the precursor of an immunoglobulin light chain containing NH2-terminal pyroglutamic acid.

Analyses of amino-acid sequences of the total cell-free products programmed by the mRNA of MOPC-104E gamma light (L)-chain show that over 95% of the products have sequences of a distinct protein that correspond to the L-chain precursor. In this precursor an extra piece is coupled to the NH2-terminus of the mature L-chain. Analyses of products labeled with [3H]alanine, [3H]leucine, and [3H]proline demonstrate that the extra piece is composed of at least 18 residues. Analyses of [35S]methione-labeled product indicate that the extra piece may contain an additional NH2-terminal methionine, which is detected in about 10% of the molecules. Partial recovery of the NJ2-terminal methionine (alanine, leucine, and proline are recovered in yields close to theoretical, greater than 95%) suggests that it is the initiator methionine, which is known to be short lived in eukaryotes due to rapid hydrolysis. Thus, the extra piece seems to be 19 residues in length, and it contains one methionine at the NH2-terminus, three alanines at positions 2, 12, and 17, and five leucines at positions 6, 8, 10, 11, and 13. The close gathering of leucine residues, as well as their abundance (26%), suggest that the extra piece would be quite hydrophobic. Hydrophobicity seems to be a general property of the extra piece, since similar clusters of leucine were found in the precursors of 3 KL-chains (Burstein, Y. & Schechter, I. (1976) Biochem. J. 157, 145-151). The NH2-terminus of the mature MOPC-104E gamma L-chain is blocked by pyroglutamic acid. The fact that in the precursor a peptide segment precedes this NH2-terminus establishes that pyroglutamic acid is not the initiator residue for synthesis of the L-chain. Apparently, the pyroglutamic acid is formed by cyclization of glutamic acid or glutamine during cleavage of the extra piece to yield the mature L-chain.     

Sequence of a mouse germ-line gene for a variable region of an immunoglobulin light chain.

We have determined the sequence of the DNA of a germ-line gene for the variable region of a mouse immunoglobulin light chain, the VlambdaII gene. The sequence confirms that the variable region gene lies on the DNA separated from the constant region. Hypervariable region codons appear in the germ-line sequence. A sequence for the hydrophobic leader, 19 amino acids that are cleaved from the amino terminus of the protein, appears near, but not continuous with, the light chain structural sequence: most of the leader sequence is separated from the rest of the gene by 93 bases of untranslated DNA.     

neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.

The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect.     

Amino acid sequence of the first constant region domain and the hinge region of the delta heavy chain of human IgD.

We have determined the amino acid sequence of the first constant (C) region domain (C delta 1) and the hinge region of the delta heavy chain of human IgD WAH and also the sequence of the adjacent COOH-terminal portion of the variable (V) region, including the JH region. Together with the sequence of the Fc fragment already reported, this establishes the complete amino acid sequence of the C region of the human delta chain and confirms the presence of three C region domains in human IgD. Although the CH1 domains of the five classes of human heavy chains have the expected degree of homology (approximately 30%), the homology of the C delta 1 domains of the human and mouse chains is less than that exhibited by the CH1 domains of other pairs of human and mouse heavy chains. The hinge region of the human delta chain has an unusual structure; the NH2-terminal half has four (or five) GalN oligosaccharides attached, whereas the COOH-terminal half lacks carbohydrate, is dissimilar in sequence, and has a high charge. A computer search verified that the GalN-rich segment has a high degree of identity in sequence with the middle portion of the human C mu 2 domain and that the high-charge segment is related to the same sequence. We propose that the two segments of the human delta hinge have a common evolutionary origin and arose by duplication and independent mutation of a hinge exon derived from the ancestral gene for the C mu 2 domain.     

Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.     

Mice completely suppressed for the expression of immunoglobulin kappa light chain.

Complete suppression of expression of immunoglobulin kappa light chain was achieved by injecting female mice from birth with a mixture of antisera against the mu heavy chain and kappa light chain (anti-mu and anti-kappa). Then their offspring were injected with anti-kappa from birth. This resulted in stable suppression as long as anti-kappa injections were continued. kappa light chain was not detectable either in serum or at the cellular level. The number of B cells in spleen and the concentration of immunoglobulin classes and subclasses in serum were normal. The normal levels were achieved by a compensating enhancement of lambda light chain expression. Analysis of the light chains of immunoglobulins secreted by spleen cells from suppressed mice after liposaccharide stimulation by two-dimensional gels showed lambda chain to have a limited heterogeneity. Primary responses to dinitrophenol, influenza strain A, and keyhole limpet hemocyanin were drastically affected, whereas secondary responses appeared to be quite normal, suggesting a surprisingly large potential repertoire.     

Complete sequence of a chicken lambda light chain immunoglobulin derived from the nucleotide sequence of its mRNA.

Recombinant cDNA plasmids have been constructed from chicken spleen poly(A)-containing RNA. Two clones have been selected and provide the sequence determination of a chicken lambda immunoglobulin light chain: they include the complete variable, constant, and 3' untranslated regions of the chicken lambda light chain mRNA and part of the leader sequence. Comparison of the chicken light chain constant region with both human and mouse lambda constant sequences indicates 61% homology at the amino acid level. Unexpectedly, the chicken variable sequence is 53-63% homologous to human variable sequences when it is compared to the various lambda subgroups and only 42% homologous to the mouse V lambda 1 sequence. The degree of homology between the variable regions of these three species does not easily correlate with their phylogenetic relationship.     

Intervening sequences divide the gene for the constant region of mouse immunoglobulin mu chains into segments, each encoding a domain.

To elucidate the structure of the gene for the constant region of immunoglobulin mu chains, we have cloned a 9.9-kilobase-pair fragment of mouse DNA bearing a gene for the constant region of the mu chain (C mu gene) from an IgM-secreting mouse plasmacytoma. The sequence around this gene has apparently undergone somatic rearrangement; the gene occurs in an EcoRI restriction endonuclease fragment of a different size from that in embryo or liver DNA and no C mu-bearing fragment of embryo size remains in the plasmacytoma. The cloned sequence lacks a variable region gene; hence, if this C mu gene is active, its position within the clone indicates that the gene for the variable region of a heavy chain (VH gene) must be more than 3.7 kilobase pairs away. The C mu gene is divided by three intervening sequences into four coding segments, each of which encodes one of the domains (homology units) of the polypeptide. The nucleotide sequence coding for amino acids near the V-C junction is not present within the C mu clone or clones bearing homologous embryonic VH genes. This suggests that an immunoglobulin heavy chain, in common with light chains, is encoded not only by a V and C gene, but also by an independent joining region (JH) gene.     

Mouse IgA heavy chain gene sequence: implications for evolution of immunoglobulin hinge axons.

The complete nucleotide sequence of the gene and mRNA coding for the constant (C) region of the secreted form of the BALB/c mouse IgA immunoglobulin alpha heavy (H) chain has been determined. As in other immunoglobulins, the three C region domains of the alpha protein, C alpha 1, C alpha 2, and C alpha 3 are coded in separate exons. However, the hinge region of C alpha is not coded on a separate exon as it is in other hinge-containing immunoglobulins. Instead, the alpha hinge is coded as a 5' extension of the C alpha 2 exon, and we suggest that it may have evolved by duplication leading to incorporation of an acceptor RNA splice site into the coding portion of the C alpha 2 exon. Extensions of this concept could provide an explanation for duplications in the human alpha 1 chain.     

Complete covalent structure of a human immunoglobulin D: sequence of the lambda light chain.

We have determined the amino acid sequence of the lambda light chain of human IgD WAH. Together with the sequence of the delta heavy chain already reported, this establishes the complete covalent structure of a human immunoglobulin D. The sequence determination was greatly aided by our ability to use high-pressure liquid chromatography to purify large peptides, including one large fragment extending from Ser-81 through the carboxyl terminus. The IgD molecule is a four-chain monomer of Mr approximately equal to 176,000; it consists of two lambda chains (each of 214 residues, Mr = 22,893) and two delta chains (each of 512 residues, Mr approximately equal to 65,000, including carbohydrate), and, unlike murine IgD, it contains two C delta 2 domains. A computer-assisted search using the J (joining) segment of the WAH lambda chain as a test piece showed a close evolutionary relationship of human and mouse J lambda regions and suggested that germ-line J lambda genes in the two species are very similar if not identical. DNA segments encoding J lambda, J kappa, and JH appear to have had a common evolutionary origin, and, surprisingly, JH seems closer to J lambda than does J kappa.     

Variable and constant parts of the immunoglobulin light chain gene of a mouse myeloma cell are 1250 nontranslated bases apart.

A DNA fragment carrying an immunoglobulin gene coding for both variable (V) and constant (C) regions of a mouse lambda light chain was enriched about 15-fold from a total endonuclease EcoRI digest of a plasmacytoma (HOPC 2020) DNA by preparative agarose gel electrophoresis. The DNA fraction was used for cloning of a lambda chain gene in the phage lambdagtWES vector. After screening [Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182] of about 70,000 plaques, each arising from an independent transfection event, we isolated one clone (Ig 303) that contained both a Vlambda and a Clambda DNA sequence. Electron microscopy of R-loops formed between the cloned DNA and purified lambda chain mRNA (HOPC 2020) revealed that the Vlambda and Clambda DNA sequences are separated by a 1250-base DNA fragment.