Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.     

Sequence of a human immunoglobulin gamma 3 heavy chain constant region gene: comparison with the other human C gamma genes.

We report the first and complete nucleotide sequence of a human gamma 3 heavy chain constant region gene (C gamma 3). This gene displays the same organization than the others C gamma genes and exhibits normal RNA splice and polyadenylation sites. A comparison of its primary sequence with those of C gamma 1, C gamma 2 and C gamma 4 genes confirms the high degree of homology (95%) of the human family in both coding and non-coding regions, and the divergence of the hinge region. The C gamma 3 gene we sequenced codes for a Gm(b) gamma 3 chain (EZZ). Comparison with other known protein sequences reveals that only two specific aminoacids are involved in the Gm(b) and Gm(g) allotypes, which suggests an important part of the spatial configuration in the allotypic specificities.     

The physical organization of the human immunoglobulin heavy chain gene complex.

Two dimensional DNA electrophoresis (2D-DE) was used to map the variable (VH) region of the human heavy chain immunoglobulin gene cluster. Seventy-six VH gene segments were mapped to specific SfiI, BssHI and NotI fragments by 2D-DE. We have determined that a common insertion/deletion polymorphism of 80 kb, involving three VH gene segments, occurs in the VH region. The physical map suggests that the evolution of the human IGH gene complex involved duplication of blocks containing different VH families. This physical map will allow comparison of the usage of VH loci in human ontogeny with their proximity to the CH region. Knowledge of the germline repertoire of a particular DNA source studied in essential as the number of the dispersed VH gene segments of VH families, especially of the VH5 family, is variable. 2D-DE, as illustrated here for the IGH gene cluster, has general application in the development of large scale physical maps of gene and repeat families.     

Organization of human immunoglobulin heavy chain diversity gene loci

The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9-kb repeating sequences. We determined the nucleotide sequence of a 15-kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9-kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12-nucleotide spacer signals. Most of the somatic DH sequences found in the published VH-DH-JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR-DH or DH-DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.     

Disulphide bridges of the heavy chain of human immunoglobulin G2

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.     

Stepwise activation of the immunoglobulin mu heavy chain gene locus.

The immunoglobulin heavy chain (IgH) gene locus spans several megabases. We show that IgH activation during B-cell differentiation, as measured by histone acetylation, occurs in discrete, independently regulated domains. Initially, a 120 kb domain of germline DNA is hyperacetylated, that extends from D(FL16.1), the 5'-most D(H) gene segment, to the intergenic region between Cmu and Cdelta. Germline V(H) genes were not hyperacetylated at this stage, which accounts for D(H) to J(H) recombination occurring first during B-cell development. Subsequent activation of the V(H) locus happens in at least three differentially regulated domains: an interleukin-7-regulated domain consisting of the 5' J558 family, an intermediate domain and the 3' V(H) genes, which are hyperacetylated in response to DJ(H) recombination. These observations lead to mechanisms for two well-documented phenomena in B-cell ontogeny: the sequential rearrangement of D(H) followed by V(H) gene segments, and the preferential recombination of D(H)-proximal V(H) genes in pro-B cells. We suggest that stepwise activation may be a general mechanism by which large segments of the genome are prepared for expression.     

c-myc Gene rearrangements involving gamma immunoglobulin heavy chain gene switch regions in murine plasmacytomas

In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

Gene conversion and polymorphism: generation of mouse immunoglobulin gamma 2a chain alleles by differential gene conversion by gamma 2b chain gene

We have determined the complete nucleotide sequence of the C57BL/6 allele of the mouse immunoglobulin gamma 2a chain gene. A comparison with the BALB/c gamma 2a gene for 1912 nucleotides reveals that the two alleles exhibit extensive divergence, since there are 138 single-base-pair differences and 8 insertions or deletions. We have compared the two gamma 2a alleles with the two corresponding gamma 2b alleles, which differ in only 12 positions. It appears that among the 134 differences between the two gamma 2a alleles, 70 are at positions where gamma 2a and gamma 2b are identical in the BALB/c haplotype and 54 are at positions where gamma 2a and gamma 2b are identical in the C57BL/6 haplotype. All these results suggest that nonreciprocal gene conversion between nonallelic genes can introduce sequence homogeneity in linked genes and can generate extensive divergence and polymorphism in allelic genes. We suggest that the gamma 2a and gamma 2b gene ancestors freely diverged after duplication, and that the conversion events were promoted by a deletion shortening the distance between the two loci.     

Human immunoglobulin heavy chain A2 gene allotype determination by restriction fragment length polymorphism.

The human immunoglobulin heavy chain alpha 2 genes have two allelic forms or allotypes called A2m(1) and A2m(2). Previously, these allotypic markers have only been distinguishable by serology. Studies of the alpha 2 genes, however, show that it is possible to differentiate between the allotypes by restriction enzyme site polymorphisms, both in the protein coding regions and in flanking regions. These polymorphic sites have been used to determine the alpha 2 allotypes of several human DNAs.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Organization of the constant-region gene family of the mouse immunoglobulin heavy chain

We cloned overlapping DNA segments that encompass the region from the immunoglobulin JH segments to the C gamma 3 gene of BALB/c mouse. We have now cloned the entire region (about 200 kilobases) of the constant-region gene family of the immunoglobulin heavy chain, the organization of which is 5'-JH-6.5 kb-C mu-4.5 kb-C delta-55 kb-C gamma 3-34 kb-C gamma 1-21 kb-C gamma 2b-15 kb-C gamma 2a-14 kb-C epsilon-12 kb-C alpha-3'. Using these cloned DNAs, we have characterized several structural features of the constant-region gene loci. There are no other J region segments except for those at the 5' side of the C mu gene. The S region is 5' to each CH gene except for the C delta gene, and the nucleotide sequences of the S region share some homology. There is no reasonably conserved pseudogene. There are at least two species of reiterated sequences scattered in these loci. Cloning and Southern blot hybridization analyses indicate that the general organizations of the heavy-chain gene loci of BALB/c and C57BL/6 mice, which have many different serological markers, are fundamentally similar but different in the lengths of S regions. Restriction enzyme cleavage maps of the whole constant-region gene loci were constructed with respect to eight restriction endonucleases.     

Primate immunoglobulin heavy chain constant gamma genes: an hypothesis of their evolution

Four Immunoglobulin gamma heavy chain isotypes are present in humans; the true phylogenetic relationship between the genes are not known because of the complex concerted evolution of the Ig multigene locus. Here we present data obtained from Southern blot analysis of the gamma genes in several primate species including prosimians (Lemur catta), New World Monkeys (Saguinus oedipus) and Old World Monkeys (Cercopitecus aethiops andMacaca fascicularis). Our data show the presence of a single IgG gene inLemur and probably inS.l oedipus, and of multiple genes in the two Cercopithecinae. These findings suggest that a single IgG gene was present in the ancestor of primates: we suppose that this IgG gene was later duplicated several times during primate evolution.