Analysis of two IgM isotypes in Atlantic salmon and brown trout

Atlantic salmon (Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CmuA and CmuB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout (Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CmuA and CmuB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CmuB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cmu subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.     

The impact of ancestral tetraploidy on antibody heterogeneity in salmonid fishes

Summary: The immunoglobulin heavy chain locus in teleost fish is structurally similar to that in mammals, comprising a series of variable gene segments upstream of two constant region genes coding for IgM and IgD. Atlantic salmon have been shown to possess two distinct heavy chain loci, related to the tetraploid ancestry of this fish family. The nature (and results) of the evolutionary processes following the tetraploidization event are the focus of this review. Salmonid fish did not return quickly to a diploid state, but are still in the process of re-establishing disomic inheritance. Thus, a specific locus in one species may still be endowed with four alleles, while it may have been converted to a pair of isoloci in another species. Analyses of immunoglobulin heavy chain genes in Atlantic salmon (Salmo salar) have strongly indicated that the ancestral heavy chain locus was subjected to tetrasomy throughout the radiation of the genera Oncorhynchus and Salmo, and that disomic inheritance was established in the Salmo lineage in the comparatively recent past. The introduction of disomic inheritance at these loci has resulted in two subsets of IgM and IgD heavy chains in Atlantic salmon.     

Cloning and structural analysis of IgM (mu chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA ( chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.     

Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.     

Characterization of the gene for the membrane and secretory form of the IgM heavy-chain constant region gene (C mu) of the cow (Bos taurus).

Our present understanding of the evolution of immunoglobulins is derived from a few vertebrate species. In order to obtain additional information on the development of the humoral immune system, we cloned and determined the nucleotide sequence of the bovine cDNA and genomic IgM heavy-chain constant region gene (C mu). The gene contains four constant region domain-encoding exons (CH1 to CH4) and two exons encoding the transmembrane domain (TM1, TM2), expressed in the membrane-bound receptor form of the IgM. The sequence of a cDNA clone encoding the 3' portion of the membrane form of the mu-chain revealed that the TM1 exon is spliced to the CH4 exon, as occurs in other mammals. Comparison of deduced amino acid sequence data from different vertebrates revealed a high similarity to sheep C mu (88%) and a lower degree of similarity to pig (62%), rat (62%), rabbit (58%) human (56%), hamster (55%), mouse (54%), chicken (28%) and horned shark (22%) C mu.     

Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern.

Rabbit antibodies to Atlantic cod (Gadus morhua L.) immunoglobulin were affinity purified and used to screen cDNA libraries from spleen and head kidney mRNA. cDNA clones for both the secretory and membrane-bound heavy (H) chain were isolated, the nucleotide and deduced amino acid sequences of which are reported here. Comparisons of the cod secretory H chain amino acid sequence show 24%, 27%, 30% identity to the mu chain of Mus, Xenopus and Ictalurus, respectively. The highest degree of identity was observed in the CH4 domain. The cDNA encoding the transmembrane form shows a novel splicing pattern where the TM1 exon is spliced directly onto the CH3 domain and not to the CH4 domain as in other animal groups. Southern blot analyses with VH and C probes on genomic DNA from cod erythrocytes indicate that there is a unique C gene but several V genes in the cod immunoglobulin H chain locus.     

Structure and organization of the T cell receptor alpha chain genes in Atlantic salmon

A cDNA fragment of the T cell receptor (TCR) alpha chain mRNA in Atlantic salmon (Salmo salar) was amplified by PCR and used as a probe to isolate a full-length clone from a leukocyte cDNA library. Additionally, a genomic lambda clone comprising the TCR alpha chain constant region (Calpha) gene and flanking regions was isolated and partially sequenced. The Calpha gene consists of three exons corresponding to the immunoglobulin (Ig) domain, the hinge region and the transmembrane peptide/cytoplasmatic tail, and two exons corresponding to the untranslated tail of the mRNA. Remnants of a transposase gene and a partial duplication of the Calpha gene were found nearby the intact gene. One J segment was found 1.5kb upstream of the Calpha gene. Twenty-six other J elements were identified among cDNA fragments covering the V/J/Calpha junction. Representatives of five Valpha gene families were identified by PCR amplification of genomic DNA fragments. PCR amplification of Calpha fragments from another individual revealed a slightly different Calpha gene which most likely represents an allelic variant.     

The major histocompatibility class II alpha chain in salmonid fishes

In this study the characterisation of the Atlantic salmon (MhcSasa-DAA) and rainbow trout (MhcOnmy-DAA) class II alpha chain cDNA sequences is presented. The DAA sequences from these two salmonid species showed a high degree of similarity, although the Onmy-DAA^*03 cDNA sequence differed in the cytoplasmic region. Interestingly, the Onmy-DAA^*02 sequence has lost the second cysteine in the alpha-1 domain. However, another cysteine is present in this sequence 7 positions downstream of the cysteine which is substituted for a leucine. Despite a thorough search, only a single locus of expressed class II alpha chain sequences was identified in both salmonid species. Amplification by PCR and sequencing of the alpha-1 domain from genomic DNA of three Atlantic salmon, identified four different variants assumed to have derived from this single locus. Two of these variants originated from one individual and are likely functional alleles.     

Sequence analysis of porcine immunoglobulin light chain cDNAs.

A porcine cDNA library was constructed using poly(A)+ RNA isolated from the spleen of an adult Minnesota miniature swine. Screening the library with antisera specific for porcine immunoglobulin light chains resulted in the selection and isolation of two recombinant clones, PLC18 and PLC3, which encode for kappa and lambda light chains, respectively. These cDNAs contain sequence information for a portion of the variable region and all of the constant region. The lengths of the constant regions are 105 amino acids for lambda and 108 amino acids for kappa. The deduced amino acid sequences of porcine immunoglobulin light chains share a high degree of homology with similar sequences from other species in both the fourth framework region and the constant region.     

Molecular cloning of the cDNA encoding the constant region of the immunoglobulin A heavy chain (Cα) from a marsupial: Trichosurus vulpecula (common brushtail possum)

A cDNA encoding the brushtail possum immunoglobulin A heavy chain constant region (Cα) was isolated by screening a mesenteric lymph node cDNA library with a porcine Cα exon 3 probe. The larger of the two positive clones isolated (Tv4a) consisted of 1325 bp of possum cDNA that included an open reading frame of 1191 bp. Its deduced amino acid sequence had a high degree of sequence identity with known eutherian Cα sequences. This clone appears to encode the entire possum IgA heavy chain constant region. The possum Cα sequence had a nucleotide sequence identity of 57.7% with porcine Cα, 51% with mouse Cα, 46.7% with dog Cα and 45.9% with human Cα2. The corresponding amino acid identities were 46.7, 45.6, 49.4 and 49%, respectively.     

Analysis and characterization of the expression of the secretory and membrane forms of IgM heavy chains in the pufferfish, Takifugu rubripes

We investigated the structure and expression of immunoglobulin genes in the pufferfish, Takifugu rubripes, a highly prized and economically important fish species. The cDNA fragment that partially encodes the constant region of the IgM heavy chain was isolated in these animals by RACE using degenerate primers after which it was used as a probe for screening IgM heavy chains in a fugu splenic cDNA library. The structural feature of the constant region of fugu sIgM was found to consist of four constant domains (CH1 to CH4), while mIgM was shown to contain a deletion of the CH4 domain, and its transmembrane domain was directly spliced to the CH3 domain as found in other teleosts. This feature may be common to all teleosts. In addition, five VH genes isolated in this study fell into two families based on their variability. Analysis of genomic sequences from the fugu genomic database also showed that there are only two VH families in the genome. The IgM gene was preferentially expressed in presumptive lymphoid tissues. Moreover, in situ hybridization revealed that large numbers of IgM positive cells were widely distributed throughout the spleen, head kidney, kidney, and thymus, confirming that these tissues were major sites of antibody production in fish. The expressions of IgM in the mucosal organs such as the skin, gills, and intestine suggest that they, too, contribute to humoral immunity in aquatic animals. The expression of IgM mRNA in the early development stages of this fish suggests that its larval form possesses a protective defense mechanism against foreign invaders.