Mechanisms of antibody diversity: Multiple genes encode structurally related mouse κ variable regions

The complete amino acid sequences of the variable regions of three mouse Vkappa-21 kappa chains (A22, T111, and CB101) and one partial sequence (B32) have been determined and are compared to four previously reported Vkappa-21 variable regions. These eight kappa variable region sequences have, with the exception of an amide difference at residue 1, identical amino-terminal 23-residue sequences, all are of the same length, and all have extensive amino acid sequence homology throughout the variable region. When these eight variable regions are grouped by sequence homology, five different groups (Vkappa-21A, B, C, D, and E) are present whose members share common sets of amino acids within a group. Three groups of similar homology each contains at least two members (M63 and AB22 in Vkappa-21B; M321 and T124 in Vkappa-21C; and M70 and B32 in Vkappa-21A). The repetition of these five characteristic subgroup sequences in this relatively small sample indicates that these subgroups are isotypes which are controlled by separate germline genes. It is unlikely that these sequences could have been randomly somatically generated in different animals from a single germ-line gene (parallel mutation). Although a limited number of comparisons are available, the sequence differences within the Vkappa-21A, B, and C isotypes are limited to complementarity-determining regions and may have resulted from somatic mutations. The kappa chains comprising the Vkappa-21 isotypes offer a unique opportunity to compare the genetic interpretations of the primary amino acid sequence data with the nucleic acid hybridization data.     

The human Thy-1 gene: structure and chromosomal location.

The human Thy-1 gene has been isolated and sequenced and compared to the rat and mouse Thy-1 genes. All three genes are organized in the same way: one exon encoding the majority of the signal peptide, another encoding the transmembrane segment, and a third encoding the remainder of the protein. One major structural difference between the human and rodent Thy-1 glycoproteins is that the former contains two instead of three glycosylation sites. RNA blot analysis of a human T-cell line expressing the T3 complex showed an absence of Thy-1 mRNA, excluding the possibility that Thy-1 represents one of the component chains of T3. The structural gene for human Thy-1 was localized to the long arm of chromosome 11 by nucleic acid hybridization to genomic DNA isolated from somatic cell hybrids.     

Organization of kappa light chain genes in germ-line and somatic tissue.

We studied the organization of the kappa light chain genes in germ-line (sperm) and somatic (embryo) tissues. We constructed a plasmid containing a DNA insert coding for the kappa chain MOPC 167 and used the Southern blotting technique to determine the organization of kappa variable and constant region genes. In the haploid genome of the mouse there is only one constant region gene detectable and it has the same organization in sperm and embryo DNAs. There are several variable region genes in sperm and embryo that are related to the Vk167 gene. The organization of the V genes in sperm and embryo DNAs is identical. These results show that there is no rearrangement of variable region genes (or "minigenes") during early embryogenesis.     

Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene.

Immunoglobulin kappa chain gene formation involves site-specific somatic recombination between one of several hundred germ-line variable region genes and a joining site (or "J segment") encoded close to the constant region gene. We have cloned and determined the nucleotide sequence of major portions of the recombination region of the mouse kappa gene and discovered a series of five such J segments spread out along a segment of DNA 2.4 kilobases from the kappa constant region gene. These J segments encode the 13 COOH-terminal amino acids of the variable region, probably including amino acids involved in the antigen combining site and in heavy/light chain contacts. The J segments also display striking sequence homology to one another in both their coding and immediately flanking sequences. Major elements of a short palindrome--CAC(TA)GTG--are preserved adjacent to the recombination sites of both variable and J region genes and constitute inverted repeats at both ends of the sequences to be joined. These palindromes can be written as a hypothetical stem structure that draws variable and J regions together, providing a possible molecular basis for the DNA joining event. Four of the J segments that we have discovered encode amino acid sequences already found in myeloma proteins. By altering the frame of recombination, we can account for additional light chain amino acid sequences, suggesting that the V/J joining event might generate antibody diversity somatically both by using different combinations of variable and J region genes and by using alternative joining frames.     

Deletions of kappa chain constant region genes in mouse lambda chain-producing B cells involve intrachromosomal DNA recombinations similar to V-J joining.

We isolated and characterized the germ-line counterpart of a DNA segment designated RS (for recombining sequence), that is frequently recombined in mouse lambda light chain-producing B lymphocytes. Using Southern blot analyses of myelomas and mouse-Chinese hamster fusion cell lines, we found that RS DNA sequences are located on mouse chromosome 6, evidently more than 15 kilobases downstream of the kappa light-chain locus. We find that a typical recognition site for Ig gene recombination is situated within germ-line RS sequences near the recombination points observed in at least two lambda chain-producing cell lines. This represents a complete and functional Ig recognition site that is not directly associated with Ig genes. We also characterized a recombined RS segment isolated from the cell line BM18-4.13.9. This recombined segment has a variable region kappa light chain gene (V kappa) joined directly to RS sequences. Our results suggest that the deletion of the kappa light chain constant region (C kappa) exon in many lambda chain-producing B cells is the result of RS recombination and that C kappa deletion may be mediated by the same processes as antibody gene V-J joining (J = joining segment gene). We discuss the potential biological significance of RS DNA recombination in B-cell maturation.     

Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.

We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.     

Polymorphic gene for human carbonic anhydrase II: a molecular disease marker located on chromosome 8.

A panel of 28 mouse-human somatic cell hybrids of known karyotype was screened for the presence of the human carbonic anhydrase II (CA II) gene, which encodes one of the three well-characterized, genetically distinct carbonic anhydrase isozymes (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1). The human and mouse CA II genes can be clearly distinguished by Southern blot analysis of BamHI-digested genomic DNA with a mouse CA II cDNA hybridization probe. The two major hybridizing fragments in mouse were 15 and 6.0 kilobase pairs, and in human they were 15 and 4.3 kilobase pairs. Analysis of the somatic cell hybrids by this technique identified those containing human CA II gene sequences. Segregation analysis of the molecular marker and chromosomes in cell hybrids indicated a clear correlation between the presence of chromosome 8 and the human CA II gene (CA2). This finding provides the second polymorphic marker for human chromosome 8 and, moreover, a molecular disease marker, because human CA II deficiency has recently been linked to an autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification.     

Leukocyte and fibroblast interferon genes are located on human chromosome 9.

At least eight leukocyte interferon genes (IFL) and the single fibroblast interferon gene (IFF) have been located on chromosome 9 in humans. In somatic cell hybrids of human and mouse cells containing a normal complement of mouse parental cell chromosomes but reduced numbers of human chromosomes, the human leukocyte and fibroblast interferon DNA sequences were present only when human chromosome 9 was also present.     

Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family.

The human type II collagen gene, COL2A1, has been assigned to chromosome 12, the type III gene, COL3A1, to chromosome 2, and one of the type IV genes, COL4A1, to chromosome 13. These assignments were made by using cloned genes as probes on Southern blots of DNA from a panel of mouse/human somatic cell hybrids. The two genes of type I collagen, COL1A1 and COL2A1, have been mapped previously to chromosomes 17 and 7, respectively. This family of conserved genes seems therefore to be dispersed throughout the genome.     

Assignment of the gene coding for the T3-delta subunit of the T3-T-cell receptor complex to the long arm of human chromosome 11 and to mouse chromosome 9.

The gene encoding the 20-kDa glycoprotein of the T3-T-cell receptor complex (T3-delta chain) has been mapped to human chromosome 11 by hybridization of a T3-delta cDNA clone (pPGBC#9) to DNA from a panel of human-rodent somatic cell hybrids. In Southern blotting experiments with DNAs of somatic cell hybrids that contained segments of chromosome 11, we were able to assign the T3-delta gene to the distal portion of the long arm of human chromosome 11 (11q23-11qter). By use of a newly developed cDNA clone (pPEM-T3 delta) that codes for the murine T3-delta chain, the mouse T3-delta gene was mapped on chromosome 9. The importance of the T3-delta map position and its relationship to the other genes on the long arm of human chromosome 11 and to those on mouse chromosome 9 is discussed.     

Homologous genes for enolase, phosphogluconate dehydrogenase, phosphoglucomutase, and adenylate kinase are syntenic on mouse chromosome 4 and human chromosome 1p

It is possible to generate interspecific somatic cell hybrids that preferentially segregate mouse chromosomes, thus making possible mapping of mouse genes. Therefore, comparison of the linkage relationships of homologous genes in man and mouse is now possible. Chinese hamster x mouse somatic cell hybrids segregating mouse chromosomes were tested for the expression of mouse enolase (ENO-1; EC 4.2.1.11, McKusick no. 17245), 6-phosphogluconate dehydrogenase [PGD; EC 1.1.1.44, McKusick no. 17220], phosphoglucomutase-2 (PGM-2; EC 2.7.5.1, McKusick no. 17190), and adenylate kinase-2 (AK-2; EC 2.7.4.3, McKusick no. 10302). In man, genes coding for the homologous forms of these enzymes have been assigned to the short arm of human chromosome 1. Analysis of 41 primary, independent, hybrid clones indicated that, in the mouse, ENO-1 and AK-2 are syntenic with PGD and PGM-2 and therefore can be assigned to mouse chromosome 4. In contrast, they were asyntenic with 21 other enzymes including mouse dipeptidase-1 (DIP-1, human PEP-C; EC 3.4.11.(*), McKusick no. 17000) assigned to human chromosome arm 1q and mouse chromosome 1. Karyologic analysis confirmed this assignment. These data demonstrate that a large autosomal region (21 map units in the mouse and 51 map units in the human male) has been conserved in the evolution of mouse chromosome 4 and the short arm of human chromosome 1. Identification of such conserved regions will contribute to our understanding of the evolution of the mammalian genome and could suggest gene location by homology mapping.     

The Organization and Diversity of Immunoglobulin Genes

We have used purified mouse immunoglobulin light chain mRNA and synthetic DNA which is complementary to it to assess the reiteration frequency of gene sequences corresponding to the kappa constant region of the mouse immunoglobulin light chain. These studies indicate that the constant region sequence is represented only two to three times per haploid mouse genome, a finding that rules out a simple stringent germ line mechanism which would require the constant region sequence to be represented hundreds if not thousands of times. Hybridization studies involving (125)I-labeled myeloma light chain mRNA yield interesting results which may eventually permit us to distinguish between the remaining somatic mutation and recombinational germ line hypotheses. These results reveal a major component of relatively unique frequency and a minor component with a reiteration frequency of approximately 30 to 50 copies per haploid genome. As discussed, these results do not permit us to distinguish unambiguously between a germ line model and a type of somatic mutation model that permits germ line genes corresponding to each kappa subgroup. The results do, however, clearly rule out the existence of thousands of variable region sequences so closely related to the MOPC-41 V-region as to permit extensive stable cross-hybridization.     

Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the beta subunit of nerve growth factor.

DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the beta subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.     

Complementation of gene deletions by cell hybridization.

Overlapping deletions in chromosome 7 of the mouse are responsible for activity deficiencies of various liver-specific enzymes, including tyrosine aminotransferase (TAT). In an effort to elucidate the nature and type of action of the deleted genes, somatic cell hybridization experiments were carried out. Enzyme-deficient liver cells of homozygous mutant mice or normal liver cells of control newborn mice were hybridized with 2S Faza rat hepatoma cells and the hybrid cell colonies were analyzed for TAT activity, The results show the presence of inducible mouse TAT activity in mutant-2S Faza hybrid cells, thereby excluding the possibility that the structural gene for TAT is included in the gene sequences deleted in the mutants. Furthermore, determinations of mouse glucose-6-phosphate isomerase 1 as a marker eliminate chromosome 7 as the possible carrier of the TAT structural gene, which therefore appears to map on a different chromosome. The deletions interfering with normal enzyme activities apparently include genes other than the respective structural genes, namely those with essential functions in controlling the expression of the differentiated state of the liver cell.     

Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins.     

Assignment of the genes for the alpha and beta subunits of thyrotropin to different mouse chromosomes.

A series of mouse-hamster somatic cell hybrids, containing reduced numbers of mouse chromosomes and a complete set of hamster chromosomes, was used to determine the chromosomal locations of the genes for the alpha and beta subunits of mouse thyrotropin. Cloned cDNA probes for each subunit, in conjunction with Southern blot analysis of DNA treated with the restriction enzyme BamHI, allowed for assignment of the alpha-subunit gene to mouse chromosome 4 and of the beta-subunit gene to chromosome 3. Mouse alpha-subunit gene sequences always segregated with chromosome 4 (concordant in 14 hybrids) and the enzyme markers phosphoglucomutase 2 and 6-phosphogluconate dehydrogenase. Mouse beta-subunit gene sequences always segregated with chromosome 3 (concordant in 15 hybrids). Thus, the genes for at least one of the glycoprotein hormones, thyrotropin, are on different chromosomes.     

Isolation and localization of DNA segments from specific human chromosomes

Recombinant DNA techniques have been combined with somatic cell genetic methods to identify, isolate, and amplify fragments of human DNA localized at specific regions of human chromosome 11 selected as a model system. A library of genomic DNA segments has been constructed, in λ Charon 4A bacteriophage, from the DNA of a somatic cell hybrid carrying a portion of human chromosome 11 on a Chinese hamster ovary cell background. Using a nucleic acid hybridization technique that distinguishes human and Chinese hamster interspersed, repetitive DNA, we have been able to distinguish recombinant phages carrying DNA segments of human origin from recombinant phages carrying DNA segments of Chinese hamster origin. We have isolated 50 human DNA segments thus far and have characterized 5 in detail. For each DNA segment characterized, a subsegment that carries no repetitive human DNA sequences has been identified. These segments have been used as hybridization probes in experiments that localize the DNA fragment on the chromosome. In each case an unequivocal chromosomal localization has been obtained with reference to a panel of hybrid cell clones each of which carries a deletion of a portion of the short arm of chromosome 11. At least one DNA segment has been identified which maps to each of the four regions on the short arm defined by the panel of hybrid cell clones used. The approaches described here appear to be general. They can be extended to produce a fine structure map of human chromosome 11 and other human chromosomes. This approach promises implications for human genetics generally, for the human genetic diseases, and possibly for understanding of gene regulation in normal and abnormal differentiation.     

The X chromosome in development in mouse and man

Summary In mammals, dosage compensation for X-linked genes between males and females is achieved by the inactivation of one of the X chromosomes in females. The inactivation event occurs early in development in all cells of the female mouse embryo and is stable and heritable in somatic cells. However, in the primordial germ cells, reactivation occurs around the time of meiosis. Owing to random inactivation in somatic cells, all female mice and humans are mosaic for X-linked gene function. Variable mosaicism can result in expression of disease in human females heterozygous for an X-linked gene defect.In the extra-embryonic lineages of female mouse embryos, and in the somatic cells of female marsupials, the paternally inherited X chromosome is preferentially inactivated. The X chromosomes in the egg and sperm must be differentially marked or imprinted, so that they are distinguished by the inactivation mechanism in these tissues.Initiation of inactivation of an entire X chromosome appears to spread from a single X-inactivation centre and may involve the recently discovered gene,XIST, which is expressed only from the inactive X chromosome. The maintenance of inactivation of certain household genes on the inactive X chromosome involves methylation of CpG islands in their 5' regions. Critical CpG sites are methylated at, or very close to, the time of inactivation in development.The mouse and the human X chromosomes carry the same genes but their arrangement is different and there are some genes in the pairing segment and elsewhere on the human X chromosome which can escape inactivation. Regions of homology between the mouse and human X chromosomes allow prediction of the map positions of homologous genes and provide mouse models of genetic disease in the human.     

The alpha-spectrin gene is on chromosome 1 in mouse and man.

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.