Structure of human immunoglobulin gamma genes: implications for evolution of a gene family

We have cloned five human immunoglobulin gamma genes from a fetal liver gene library. Four of them encode the known human immunoglobulin gamma chains gamma 1, gamma 2, gamma 3 and gamma 4. A fifth gamma gene seems to be a pseudogene. Nucleotide sequence determination demonstrates that the gamma 3 gene contains four separate hinge exons. Comparison of these hinge exons with those of the other gamma genes indicates that the first hinge exon is homologous to that of the pseudogene, and that the other three hinge exons are homologous to that of the gamma 1 gene, suggesting that the gamma 3 gene ancestor is a hybrid gene created by unequal crossing-over between the ancestral gamma 1 and psi gamma genes. Amplification of the gamma 1-type hinge exon probably followed to complete the gamma 3 gene. This hypothesis inevitably postulates the gene order 5'-gamma 1-gamma 3-psi gamma-3'. Cloning of overlapping chromosomal segments demonstrates that the gamma 2 gene is located 19 kb 5' to the gamma 4 gene. These analyses indicate that the human gamma-gene family has evolved by several types of DNA rearrangemet, including duplication of a complete gene; duplication of a hinge exon; and reassortment of exons by unequal cross-over between two adjacent genes.     

Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.     

Isolation and characterization of cloned human fetal globin genes.

Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clone (NY 1) has a 15.4 kb insert by virtue of an extra 1.4 kb Eco RI fragment at its 5' most end. This clone also has a Kpn I site not present in the other two suggesting it is the product of the gamma gene on the opposite chromosome. Restriction analysis of the three clones indicates that the G gamma and A gamma genes are linked on a single continuous piece of DNA and are separated by 3.5 kb and each contains at least one large intervening sequence of 0.85 kg between the Bam HI and Eco RI sites. These findings in cloned DNA provide direct evidence for linkage and organization of the gamma genes in man.     

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

Molecular cloning of rabbit gamma heavy chain mRNA.

A cDNA library of rabbit spleen mRNA was screened for immunoglobulin heavy chain sequences. In this paper we report the nucleotide sequence of two cDNA clones containing part of the constant region of the rabbit gamma heavy chain mRNA. The sequence encodes part of the CH2 domain (amino acids 268 to 340), the entire CH3 domain (amino acids 341 to 447) and the 3' untranslated region. This nucleotide sequence has been compared to the corresponding sequences of mouse gamma 1, gamma 2a and gamma 2b genes. The homologies between rabbit gamma chain gene sequence and each of the mouse gamma chain gene sequences are of the same magnitude order. This comparison shows that the CH2 domains are more homologous to each other than CH3 domains or 3' untranslated sequences. The presence of species specific nucleotide positions suggests that mouse gamma chain genes could have evolved from a common ancestor shortly after the mouse-rabbit species separation. Genomic blot analysis of rabbit liver DNA with the rabbit C gamma probes shows a limited number of related sequences, with little restriction site polymorphism between individual rabbits.     

The nucleotide sequence of a human immunoglobulin C gamma1 gene

We report the nucleotide sequence of a gene encoding the constant region of a human immunoglobulin gamma 1 heavy chain (C gamma 1). A comparison of this sequence with those of the C gamma 2 and C gamma 4 genes reveals that these three human C gamma genes share considerable homology in both coding and noncoding regions. The nucleotide sequence differences indicate that these genes diverged from one another approximately 608 million years ago. An examination of hinge exons shows that these coding regions have evolved more rapidly than any other areas of the C gamma genes in terms of both base substitution and deletion/insertion events. Coding sequence diversity also is observed in areas of CH domains which border the hinge.     

Normal T cell development is possible without ''functional'' gamma chain genes.

The T cell-specific gamma gene family is organized into four V, J and C gene segments containing clusters (gamma 1, gamma 2, gamma 3, gamma 4) in germline DNA. We found that the V, J and C elements of gamma 2 are physically linked on a stretch of 6 kb of DNA while those of gamma 3 are found within a 15-kb region. Rearrangements take place only within the clusters, explaining the rigid rearrangement patterns seen in T lymphocytes. New V gamma, J gamma and C gamma gene segments were discovered and characterized allowing the better understanding of the potential germline diversity of the gamma gene family. No correlation with T cell function, i.e. cytolytic or helper, and the type of the productive gamma rearrangement could be established. In contrast we found that functional T cell clones have been able to mature without any functional gamma chain genes.     

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.     

Organization and polymorphism of rabbit immunoglobulin heavy chain genes

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.     

gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.     

Evolution of the six horse IGHG genes and corresponding immunoglobulin gamma heavy chains

It is generally assumed that the different mammalian IgG isotypes have developed during evolution by duplications of a common ancestor gamma heavy chain constant region gene (IGHG). In contrast to other species studied so far, which express between one and four IGHG genes, the horse (Equus caballus) genome contains six IGHG genes, and it has been postulated that they all can be expressed. For determination of the evolutionary history of the six horse IGHG genes, genomic DNA and cDNA of the IGHG genes were sequenced. The structure of these genes with reference to exons and introns was determined. Comparison of the deduced amino acid sequences of the horse IGHG genes revealed the greatest divergences in the hinge regions, and in the proximal CH2 domains. A phylogenetic comparison of the amino acid sequences of the six horse IGHG genes to those of other species shows that the horse IGHG genes form a distinct cluster. This indicates that the mammalian species included in this study probably share only one common ancestor IGHG gene with the horse. The six horse IGHG genes probably then evolved by gene duplication after species separation. In addition, various segmental exchanges were found between the horse IGHG genes, which might be the result of unequal crossing over and/or gene conversion events during the evolution of the six horse IGHG genes.     

Intracisternal A-particle genes: structure of adjacent genes and mapping of the boundaries of the transcriptional unit

Adjacent intracisternal A-particle (IAP) genes were identified in two different recombinant DNA clones, gamma 81 and gamma 19. In clone gamma 81, the most common form of IAP gene was separated by 5.3 kilobases from another IAP gene that had two apparent internal deletions. The two genes were in a head-to-tail configuration. In clone gamma 19, two different types of IAP genes were separated by less than 0.5 kilobase. Blot hybridization analysis of mouse DNA demonstrated that the DNA sequence found in clone gamma 81 is identical to the in vivo configuration. Using isolated DNA fragments from clone gamma 19, we mapped the boundaries of the IAP RNA by S1 digestion of RNA-DNA hybrids and by cDNA extension. With these techniques, both the 5' end and the 3' end of the IAP RNA in two different plasmacytomas (MOPC 315 and TEPC 15) were shown to fall within the long terminal direct repeat of the IAP gene. The fragment sizes generated by S1 digestion of IAP RNAs isolated from the two tumor lines were found to differ, indicating that different IAP genes may be transcribed in these two plasmacytomas.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein

Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein.     

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.     

Concerted evolution of the mouse immunoglobulin gamma chain genes

The nucleotide sequences of the immunoglobulin heavy-chain constant region genes of mouse, C gamma 3, C gamma 1, C gamma 2b and C gamma 2a, together with that of a human equivalent C gamma 4 were compared. All the six pairs of genes within the mouse C gamma gene family contain DNA segments that exhibit marked homology, whereas no such segmental homology was found in interspecies comparisons. This result indicates that the four C gamma genes of the mouse evolved concertedly by exchanging parts of their genetic information with each other either by gene conversion or by double unequal crossing-over. Another example of such concerted evolution was found in gene regions encoding membrane domains of the mouse C gamma chains. We also searched for such segmental homologies in other mammalian C gamma gene families and found at least two more examples in man and guinea-pig. In the mouse C gamma gene family, the silent positions of an exon encoding the third domain of C gamma chains show much greater divergence in sequence than other regions, indicating that the genetic information encoded by this gene region was least scrambled during recent evolution. A phylogenetic tree constructed from the nucleotide differences of this exon demonstrates that at least two C gamma genes had already existed before mammalian radiation. Based on these results, evolution of mammalian C gamma gene families is discussed.     

Chimpanzee fetal G gamma and A gamma globin gene nucleotide sequences provide further evidence of gene conversions in hominine evolution

The fetal globin genes G gamma and A gamma from one chromosome of a chimpanzee (Pan troglodytes) were sequenced and found to be closely similar to the corresponding genes of man and the gorilla. These genes contain identical promoter and termination signals and have exons 1 and 2 separated by the conserved short intron 1 (122 bp) and exons 2 and 3 separated by the more rapidly evolving, larger intron 2 (893 bp and 887 bp in chimpanzee G gamma and A gamma, respectively). Each intron 2 has a stretch of simple sequence DNA (TG)n serving possibly as a "hot spot" for recombination. The two chimpanzee genes encode polypeptide chains that differ only at position 136 (glycine in G gamma and alanine in A gamma) and that are identical to the corresponding human chains, which have aspartic acid at position 73 and lysine at 104 in contrast to glycine and arginine at these respective positions of the gorilla A gamma chain. Phylogenetic analysis by the parsimony method revealed four silent (synonymous) base substitutions in evolutionary descent of the chimpanzee G gamma and A gamma codons and none in the human and gorilla codons. These Homininae (Pan, Homo, Gorilla) coding sequences evolved at one-tenth the average mammalian rate for nonsynonymous and one-fourth that for synonymous substitutions. Three sequence regions that were affected by gene conversions between chimpanzee G gamma and A gamma loci were identified: one extended 3' of the hot spot with G gamma replaced by the A gamma sequence, another extended 5' of the hot spot with A gamma replaced by G gamma, and the third conversion extended from the 5' flanking to the 5' end of intron 2, with G gamma replaced here by the A gamma sequence. A conversion similar to this third one has occurred independently in the descent of the gorilla genes. The four previously identified conversions, labeled C1-C4 (Scott et al. 1984), were substantiated with the addition of the chimpanzee genes to our analysis (C1 being shared by all three hominines and C2, C3, and C4 being found only in humans). Thus, the fetal genes from all three of these hominine species have been active in gene conversions during the descent of each species.      

Gene conversion in human immunoglobulin gamma locus shown by unusual location of IgG allotypes

The constant region of the gamma 1, gamma 2 and gamma 3 heavy chains of the human IgG1, IgG2 and IgG3 immunoglobulins carries antigenic determinants or G1m, G2m and G3m allotypes, which are genetic markers of these subclasses. The exceptional presence on gamma 1 and gamma 2 chains of Gm allotypes usually located on the CH3 domain of gamma 3 shows an unexpected clustering of base changes and subsequent identity of short DNA sequences in the CH3 exon of the non-allelic gamma 1, gamma 2 and gamma 3 genes. Such clusters of substitutions are not easily explained on the classical basis of point mutations. A gene conversion, which substituted a segment of the gamma 1 or gamma 2 gene with the homologous region of the non-allelic gamma 3 gene, is more likely. Other examples of possible conversion involving the gamma genes are described. The conservation or the restoration of short sequences produced by the conversion events might be related to the biological properties of the constant region of the heavy chains.     

IL-4 induces the specific rearrangement of gamma 1 genes on the expressed and unexpressed chromosomes of lipopolysaccharide-activated normal murine B cells

Small, resting, surface IgM+/surface IgD+ murine B cells undergo an Ig class switch to IgG1 or IgE after stimulation with LPS and T cell supernatants containing IL-4. To firmly establish the role of IL-4 in the directed switch recombination observed in IgG1-secreting cells, we have 1) used highly purified native IL-4 instead of T cell supernatants, 2) used resting B cells from F1 mice in which the active IgH allele was determined before culture, 3) taken advantage of the allelic differences in the restriction fragment lengths of mu, gamma 1, gamma 2b, and gamma 3 loci to determine the status of the CH genes on both the expressed and unexpressed chromosomes, and 4) used different restriction enzymes to distinguish between deletion and rearrangement of a given CH gene. Our results indicate that LPS alone induces rearrangement of the gamma 3 genes on both chromosomes whereas stimulation with LPS plus IL-4 results in deletion of gamma 3 genes and rearrangement of gamma 1 genes on both chromosomes. The studies definitively establish the role of IL-4 in directed switch recombination to the gamma 1 locus in LPS-stimulated murine B cells.