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The Journal of Immunology, 1998, 161: 6724-6732.
Copyright © 1998 by The American Association of Immunologists

Marsupial Light Chains: Complexity and Conservation of {lambda} in the Opossum Monodelphis domestica1 ,2

Julie E. Lucero, George H. Rosenberg and Robert D. Miller3

Department of Biology, University of New Mexico, Albuquerque, NM 87131


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Ig{lambda} chains in the South American opossum, Monodelphis domestica, were analyzed at the expressed cDNA and genomic organization level, the first described for a nonplacental mammal. The V{lambda} segment repertoire in the opossum was found to be comprised of at least three diverse V{lambda} families. Each of these families appears to be related to distinct V{lambda} families present in placental mammals, suggesting the divergence of these genes before the separation of metatherians and eutherians more than 100 million years ago. Based on framework and constant region sequences from full-length cDNAs and intron sequences from genomic clones, it appears that there are multiple functional J{lambda}-C{lambda} pairs in the opossum locus. The opossum J{lambda}-C{lambda} sequences are phylogenetically clustered, suggesting that these gene duplications are more recent and species specific. Sequence analysis of a large set of functional, expressed V{lambda}-J{lambda} recombinations is consistent with an unbiased, highly diverse {lambda} light chain repertoire in the adult opossum. Overall, the complexity of the Ig{lambda} locus appears to be greater than that found in the Ig heavy chain locus in the opossum, and light chains are therefore likely to contribute significantly to Ig diversity in this species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Two identical heavy chains paired with two identical light chains is the generic structure of vertebrate Ig molecules. In mammals, the Ig heavy chains (IgH)4 are encoded at a single site in the genome, but the two types of Ig light chains (IgL), {kappa} and {lambda}, are encoded at separate, unlinked loci. The use of the two IgL types can vary between species, some having a bias or preferential use of one over the other (reviewed in 1 . Mice and rabbits, for example, use predominantly Ig{kappa}, whereas horses, sheep, and cattle use primarily Ig{lambda} (1, 2, 3, 4, 5, 6). The use of one IgL type over another correlates, in general, with the overall complexity of the loci in most species. Humans, for example, have a significant amount of V{lambda} and V{kappa} diversity and use both extensively, 60% Ig{kappa}:40% Ig{lambda} (2, 7, 8, 9). Mice, on the other hand, have only three functional V{lambda} segments but a large number of available V{kappa} and have a 95% Ig{kappa}:5% Ig{lambda} ratio (7, 9). The contributions that IgL make to Ab diversity can also vary greatly between species. Humans appear to have a significant amount of light chain diversity (7, 10). In contrast, the {lambda} repertoire of cattle is restricted to a recurrent V{lambda}-J{lambda} rearrangement, even though they appear to have multiple functional V{lambda} and J{lambda} segments in their germline (5). Perhaps the most extreme case of limited contribution by light chains occurs in the camelids (camels and llamas), which produce a form of IgG lacking light chains entirely (11).

Our knowledge of the structure, diversity, and evolution of the mammalian IgL genes is based on studies of only one of the three major orders of mammals, the eutherians or "placental" mammals. To date there has been no reported IgL gene structure from either of the other two mammalian orders, the prototherians (egg laying monotremes, e.g., the platypus) or the metatherians (marsupials). The relationship of these three mammalian lineages has been a subject of continued debate over much of this century with most investigators placing the metatherians and the eutherians together as sister taxa, with the prototherians diverging earliest (12). However, more recent analysis of mitochondrial DNA supports the idea that prototherians and metatherians are sister taxa, with the eutherians splitting off first (12, 13). Possible times for the divergence of these groups range from less than 120 million years ago, during the Cretaceous Period, to possibly greater than 170 million years ago, during the Jurassic Period (14, 15). A more extensive analysis of metatherian and prototherian immunobiology provides a comparison between very distantly related mammalian species and should yield important knowledge into the evolution of mammalian immune systems.

In addition to their importance to mammalian evolution, marsupials also provide an opportunity to study mammals that are born comparatively less developed than mice or humans. Developmental immaturity combined with the lack of a placenta, which supports the transfer of maternal Ig, in most marsupial species creates unique immunological problems for metatherians (16). The opossum, Monodelphis domestica, has been established over the last decade as an important laboratory-bred marsupial for studies of many areas of comparative and biomedical research (17, 18). M. domestica are native to South America and are a member of the family Didelphidae, which contains the largest number of species within the marsupials, and Monodelphis is the most species-rich genus of the family (19). The Didelphidae are also thought to have diverged earliest from the rest of the metatheria and may contain some of the oldest extant mammalian species (20, 21).

We have begun characterizing the Ig genes of M. domestica, and we previously reported that the IgH repertoire was derived from two related group III type VH families (22). To extend this analysis to opossum IgL, we have cloned and characterized Ig{lambda}-containing cDNAs and have found the presence of at least three highly divergent V{lambda} families, the absence of bias in the V{lambda}-J{lambda} combinations, and evidence that the duplicated J{lambda}-C{lambda} pair arrangements found in placental mammals is conserved in the opossum. It appears that the genetic complexity of the M. domestica Ig{lambda} locus is greater than that for the IgH locus, suggesting that {lambda} light chains contribute significantly to the diversity of the Ig repertoire in this species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PCR amplification of Ig{lambda} sequences

A degenerate oligonucleotide (5'-CCNGGYTTYTGYTGRTACCA) complementary to the coding strand for the amino acid sequence WYQQKPG conserved in framework region 2 (FR2) of light chain V segments (also see 23 was used to amplify opossum VL fragments by anchored PCR. The target for PCR was a commercially available M. domestica spleen cDNA library constructed using the {lambda}ZAPII cloning vector (Stratagene, La Jolla, CA). The degenerate FR2 oligonucleotide was used in PCR as a reverse primer in combination with the T3 universal sequencing primer specific for a site flanking the cloning site in {lambda}ZAPII. Successful amplification was achieved using 2 mM MgCl2 and 55°C annealing temperature and Taq polymerase (Perkin-Elmer, Foster City, CA). For this study, all PCR products were cloned for sequencing or for use as probes using the pCR2.1 vector (Invitrogen, Carlsbad, CA) following the manufacturer’s recommended protocol. An oligonucleotide primer complementary to the 5' region of M. domestica C{lambda} (5'-ACCATAGGCCATGACCATGG) was paired in PCR with the T3 primer to amplify V{lambda} region segments in an unbiased manner. The spleen cDNA library described above was used as target with the conditions of 3.0 mM MgCl2 and 55°C annealing temperature.

In experiments to confirm the germline J{lambda}-C{lambda} pair arrangement, oligonucleotides for each known M. domestica J{lambda} segment (J{lambda}I, 5'-GTGTTCGGCAGTGGGACCAG; J{lambda}II, 5'-GTGTTCGGTGGTGGGACCAA; J{lambda}III, 5'-GTGTTCGGTGCTGGGACCAA; J{lambda}IV, 5'-GTGTTCGGCCGTGGGACCAG; J{lambda}V, 5'-GTGTTTGGCGGTGGGACCAA; J{lambda}VI, 5'-GTGTTCGGCGGTGGGACCAG) were paired with the C{lambda} primer described above to amplify genomic fragments. Amplifications were performed using PCR with 2 mM MgCl2 and a 60°C annealing temperature.

Blot hybridizations

All genomic M. domestica DNA used were extracted from spleen tissue using standard protocols. For Southern blot analysis, genomic DNA were cut with various restriction endonucleases following the manufacturer’s recommended conditions (see figure legends). Digested DNA were electrophoresed through 1% agarose (FMC Bioproducts, Rockland, ME) and transferred to reinforced nitrocellulose for probing (Micron Separations, Westborough, MA). Phage plaque lifts for cDNA library screening were also made using reinforced nitrocellulose. All probes used in this study were prepared as DNA inserts excised from plasmids and labeled with [32P]dCTP by the random primer method (Prime-it Kit, Stratagene). Hybridizations were done at 42°C in 50% formamide, 5x Denhardt’s solution, 5x SSC, 50 mM NaPO4 (pH 6.5), 0.1% SDS, 5 mM EDTA, and 250 mg/ml sheared salmon sperm DNA. Final wash conditions were 65°C and 0.2x SSC.

Sequencing and analysis

DNA sequencing reactions were performed using the ThermoSequenase sequencing kit (Amersham, Arlington Heights, IL), and the reactions were analyzed using an automated DNA sequencer (Perkin-Elmer ABI Prism 377 DNA sequencer). All DNA sequences reported were derived by completely sequencing both strands of each clone. Sequences were analyzed using the Sequencher 3.0 program (Gene Codes, Ann Arbor, MI), and alignments were constructed using the CLUSTAL W program (24). All phylogenetic trees shown are reconstructed from nucleotide alignments. To align the nucleotide sequences, first the amino acid translations were aligned using CLUSTAL W with minor manual corrections, then nucleotide sequences were aligned and gapped manually based on the protein alignments to retain codon positions. Based on these nucleotide alignments, trees were reconstructed using the neighbor-joining method of Saitou and Nei (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of M. domestica Ig{lambda} cDNAs

To isolate clones containing opossum Ig{lambda} sequences, fragments of V{lambda} segments were first amplified from a spleen cDNA by anchored PCR using a FR2-specific degenerate primer and then, cloned and sequenced. Four unique clones were found to be homologous to the leader, FR1, and complementarity determining region 1 (CDR1) of known mammalian V{lambda} segments (not shown). The clones varied from 175 to 184 nucleotides in length and shared from 52% to 74% nucleotide similarity to mammalian V{lambda}, but less than 40% similarity to any mammalian V{kappa} sequences (not shown). Two of the PCR-generated clones were from different V{lambda} families, based on less than 50% similarity, and were used independently to screen a cDNA phage library constructed from M. domestica spleen RNA. Three clones were identified using each probe, and all six clones were found to contain full length {lambda} light chain cDNAs containing variable and constant regions (Fig. 1Go).



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  		FIGURE 1. Nucleotide alignment of six full-length cDNA clones, including 5' and 3' untranslated regions (UTR). The starting point for the leader, FR, CDR, and constant regions are indicated by a filled circle. The cDNAs are grouped based on the similarity of V sequences from FR1 through FR3.

 
Identification of three V{lambda} families in M. domestica

The six full length cDNA sequences shown in Fig. 1Go are grouped by nucleotide similarity in the V region. The presence of at least two V{lambda} families, which have been designated opossum V{lambda}1 (clones 2c, 3c, and 4c) and V{lambda}2 (clones 7c, 10c, and 12c), is apparent in the opossum Ig{lambda} repertoire. The separation of these sequences into two V{lambda} families is based on a typically >87% similarity among sequences in the same family and <56% similarity between the families.

To rapidly screen for the presence of additional V{lambda} families, an oligonucleotide primer complementary to coding sequence near the 5' end of the C{lambda} region was paired with a primer specific for the T3 promoter sequence flanking the cloning site in the phage vector used to construct the cDNA library. This approach amplifies V domain sequences, using the spleen cDNA library as target, without bias for V{lambda} or J{lambda} sequences. The sequence of the C{lambda} primer was complementary to nucleotides 422–441 in the C{lambda} region shown in Fig. 1Go, which is a sequence common to all 6 C{lambda} regions found so far. A total of 40 unique V{lambda}-J{lambda} rearrangements were amplified from the cDNA library and then, cloned and sequenced. Of these new sequences, the majority (36 total) clearly grouped with the V{lambda}1 family, while 2 grouped with the known V{lambda}2 family (sequences 46p and 62p in Fig. 2Go). The remaining 2 clones (sequences 18p and 25p in Fig. 2Go) shared 97% nucleotide similarity to each other, but <65% similarity to any V{lambda}1 or V{lambda}2 sequences, and defined a third V{lambda} family, opossum V{lambda}3. One clone (51p in Fig. 2Go) was grouped as a V{lambda}1 member but clearly contains a FR3 from the V{lambda}2 family. Whether this clone contains a bona fide germline V segment that may have undergone gene conversion or recombination, or is an artifact of template jumping during PCR, remains to be determined.



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  		FIGURE 2. Presence of three V{lambda} families. Nucleotide alignment of the V domain sequence amplified by anchored PCR. Included for comparison are the V regions of the six cDNA clones in Fig. 1Go. Gaps in the alignment are indicated by dots and the sequences are gapped to match codon position. Roman numerals at the end of the sequences designate similar FR4 sequences.

 
An unusual feature that distinguishes the three families is the presence of consistently shorter CDR1 and CDR2 regions in the V{lambda}2 and V{lambda}3 families when compared with the V{lambda}1 members. The V{lambda}2 and V{lambda}3 segments are one codon shorter than V{lambda}1 in CDR1 and four and three codons shorter in CDR2, respectively. In addition, the CDR3 regions created by the V{lambda}-J{lambda} junction are also consistently shorter in those clones that contain rearrangements involving V{lambda}2 and V{lambda}3 family members. The length of the CDR3 does not appear to correlate with a bias in V{lambda}-J{lambda} combinations. Based on FR4 sequences, we estimate there to be at least six functional J{lambda} segments in the M. domestica Ig{lambda} locus (indicated by the Roman numerals next to the FR4 sequences in Fig. 2Go). All six J{lambda} segments can be found in rearrangements that contain a V{lambda}1 and long CDR3 regions, while four of six J{lambda} segments can be found in rearrangements that contain a V{lambda}2 or V{lambda}3 with comparatively shorter CDR3 regions. In summary, there appears to be no relationship between the combination of particular J{lambda} with specific V{lambda} segments, and the length of the CDR3 region does not associate with particular J{lambda} segments.

To estimate the number of V{lambda} gene segments present in the M. domestica genome, Southern blot hybridizations were performed using representative clones from each of the three families as probes (Fig. 3Go). A V{lambda}1 probe hybridized to an average of 20 restriction fragments in the M. domestica genome (Fig. 3GoA). This same blot was stripped and rehybridized with probes specific for the V{lambda}2 (Fig. 3GoB) and V{lambda}3 (Fig. 3GoC) families, which revealed 8 and 4 genomic fragments, respectively.



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  		FIGURE 3. Determination of the number of V{lambda} segments in the M. domestica genome by Southern blot analysis. Genomic DNA was digested with the indicated restriction enzyme, electrophoresed, blotted, and probed with a DNA fragment containing sequence that was representative of a V{lambda}1 (clone mvl-5a, not shown), V{lambda}2 (subclone of clone 46p in Fig. 2Go), or V{lambda}3 (subclone of clone 25p in Fig. 2Go). Restriction enzymes are shown as: B, BamHI; EI, EcoRI; EV, EcoRV; H, HindIII; P, PstI; S, SacI; X, XbaI.

 
Evidence for multiple J{lambda}-C{lambda} pairs in M. domestica

An alignment of the nucleotide (Fig. 2Go) or amino acid sequence encoded (Fig. 4Go) by the six full-length cDNAs revealed three distinct pairs of sequences based on FR4 and C regions. Unlike the order of the clones presented in Fig. 2Go, the six sequences in Fig. 4Go are grouped based on similar FR4 regions (amino acid positions 105–116) to illustrate the paired relationships. The FR4 regions of cDNA clones 2c and 7c are nearly identical, and there is significant similarity in the FR4 regions of clones 3c and 12c, as well as 4c and 10c. Comparison of the six C{lambda} sequences reveals identical paired patterns of similarity; in other words, cDNA clones with similar FR4 sequences share similar C{lambda} sequences. The most likely explanation for this pattern is the presence of multiple functional J{lambda} segments, each with its own C{lambda} downstream.



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  		FIGURE 4. Alignment of deduced amino acid sequences of the full-length M. domestica Ig{lambda} cDNA clones from Fig. 1Go. Sequences are paired based on similar constant region sequences.

 
To confirm the presence of multiple J{lambda}-C{lambda} pairs in the opossum genome, primers were designed to be unique for each of the six known FR4 regions (J{lambda}) and paired with the C{lambda} primer for PCR using genomic DNA as a target. PCR amplification with each FR4 primer paired with the C{lambda} primer yielded products ~1.8 kb long, which were cloned and partially sequenced. Sequences internal to the primers confirmed that the amplified fragments contained an intron with predicted splice sites flanked by J{lambda} and C{lambda} segments, and each clone had a unique restriction map (Fig. 5Go). These results confirm the presence of multiple functional J{lambda}-C{lambda} pairs in the opossum genome. A Southern blot of M. domestica genomic DNA probed for C{lambda} revealed typically six to eight fragments (Fig. 6Go) consistent with the estimate of at least six unique J{lambda} segments based on FR4 sequences and the presence of J{lambda}-C{lambda} pairs.



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  		FIGURE 5. Sequence and partial restriction map of J{lambda}-C{lambda} clones generated by PCR from genomic DNA. The complete nucleotide sequences of the introns are not shown, and the line representing the intron is not drawn to scale. Roman numerals on the left of the figure indicate the different J segment sequences. Nucleotides corresponding to the oligonucleotide sequences used as PCR primers have a double underline. The predicted splice sites flanking the intron are underlined. A consensus amino acid translation of the sequence internal to the primers is shown below the nucleotides. Restriction sites within the intron are shown as: A, ApaI; B, BstXI; D, DraII; E, EcoRI; H, HindIII; S, SmaI; Sc, SacI; X, XmaIII.

 


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  		FIGURE 6. Southern blot analysis of the opossum C{lambda} genes. Genomic DNA was digested with the indicated restriction enzyme and probed with a subcloned fragment of clone 7c in Fig. 1Go. Restriction sites are shown as: B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SacI; Sp, SpeI; X, XbaI; Xh, XhoI.

 
Phylogenetic analysis of the opossum V{lambda} families and C{lambda} regions

Pairwise comparisons of the opossum sequences with V{lambda} sequences from placental mammals revealed greater similarity between the opossum V{lambda} families and V{lambda} sequences from other species than that found between opossum V{lambda} families. To illustrate these relationships, a phylogenetic tree was constructed using opossum V{lambda} aligned to representative V{lambda} sequences from other mammals. Fig. 7GoA shows a tree based on nucleotide alignment of the FR regions of the 3 opossum V{lambda} families and the 10 human V{lambda} families. Also included in the alignment were sequences from mice, rabbits, and two artiodactyl species, cattle and sheep. There was no difference in the tree topology when CDR sequences were included in the alignments (not shown). The overall topology of the tree, or relationship among the mammalian V{lambda} sequences, is in general agreement with that reported in more extensive analysis of vertebrate VL sequences (26, 27). Mouse V{lambda}2 was excluded from the alignment because it is highly similar to mouse V{lambda}1. The rabbit and artiodactyl sequences cluster on their own branches, whereas the 2 mouse sequences and 10 human V{lambda} families are more dispersed around the tree. The opossum V{lambda} families also intersperse with the sequences from mice and humans. This result suggests that the gene duplication events that produced these families predate the evolutionary separation of mammals.



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  		FIGURE 7. Phylogenetic analysis of opossum V{lambda} and C{lambda} sequences. A, V{lambda} tree based on a nucleotide alignment of the FR sequences. Representatives of each of the 10 human V{lambda} families (VL1–10 on the tree) were taken from the VBASE database (29). The opossum V{lambda}1, V{lambda}2, and V{lambda}3 representatives are sequences 2c, 7c, and 18p, respectively, from Fig. 2Go. Sequences from the other taxa were downloaded from the GenBank database: mouse V{lambda}1 (J00590), V{lambda}x (D38129); rabbit V{lambda}2 (M27840), V{lambda}3 (M27841); cattle V{lambda}1a (U31106); sheep V{lambda}5.1 (M60441), V{lambda}5.2 (AF040918). B, C{lambda} tree based on nucleotide alignments from the opossum sequences from the six cDNA clones shown in Fig. 1Go. Sequences of other mammalian taxa and two avian species were downloaded from GenBank: chicken C{lambda} (K00678); duck C{lambda} (X82069); mouse C{lambda}1 (J00587), C{lambda}2 (J00595), C{lambda}3 (J00585), C{lambda}5 (M35582); human C{lambda}1 (X51755), C{lambda}2 (J00253), C{lambda}3 (J00254), C{lambda}6 (J03011), C{lambda}7 (M61771); rabbit C{lambda}1 (M12388), C{lambda}2 (M12761), C{lambda}4 (M12763). Mouse V{lambda}2 is very similar to mouse V{lambda}1 and was not included in the alignment. Mouse C{lambda}4 is a pseudogene and very similar to mouse C{lambda}1 and was not included in the alignment. Scale bars indicate frequency of substitutions per site.

 
Phylogenetic trees constructed from nucleotide alignments of all six C{lambda} sequences from the cDNA clones in Fig. 1Go with C{lambda} regions from other species revealed a strikingly different pattern of evolution at the C end of the opossum Ig{lambda} compared with the V end (Fig. 7GoB). In the case of C{lambda}, the duplication events appear to have occurred after speciation. The C{lambda} regions of the opossum all cluster at the end of a long branch and, likewise, the duplicated C{lambda} regions of mouse, human, and rabbit all cluster on their own branches. Two avian C{lambda} sequences were included for comparison. Mouse C{lambda}4 was excluded because it is a pseudogene and highly similar to C{lambda}1. Several relationships support the validity of this tree, including the intraspecies clustering of C{lambda} sequences previously noted (see 26 , and the common branch that the mouse surrogate light chain C{lambda}5 shares with C{lambda}1; C{lambda}5 is thought to be derived from C{lambda}1 (28).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The gray, short-tailed opossum, M. domestica, has been an important model for studies of marsupial immunobiology (30), but much work remains to develop the reagents needed to study marsupial immune systems at the level of sophistication achieved for mice and humans. Toward improving our knowledge of immunogenetics in this species, and to gain insight into the evolution of mammalian Ag receptors, we have been characterizing the opossum homologues of immunologically relevant genes including IgH, the recombination activating gene-1, and terminal deoxynucleotidyl transferase (22, 31, 32). We present here the first molecular characterization of a marsupial IgL. The {lambda} light chain repertoire of the opossum is derived from at least 3 ancient V families, which total ~30 gene segments. These V segments appear to randomly recombine with available J segments, giving a potential combinatorial diversity for opossum {lambda} comparable to that described in humans. The first important conclusion from our results is that {lambda} has been retained in the metatherian lineage. This is not unexpected given that both {lambda}- and {kappa}-like sequences have been described in all vertebrate groups, including sharks (23, 33, 34, 35, 36, 37, 38, 39).

Evolution of mammalian V{lambda}

Phylogenetic analysis of V{lambda} and V{kappa} sequences from several vertebrates revealed the presence of multiple V{lambda} groups, but only a single V{kappa} cluster, hence V{lambda} has been referred to as being "polyphyletic" compared with the V{kappa} (26, 27). In addition, phylogenetic analysis of V{lambda} sequences by Hayzer (26), Sitnikova and Su (27), and Zezza et al. (36) all generally agree that human and mouse V{lambda} families intersperse with genes from other vertebrates, while sequences from other species generally remain clustered with their phylogenetic origin (i.e., all rabbit V{lambda} clustered within a single group, all avian V{lambda} clustered within a single group, etc.). Reconstruction of a phylogenetic tree that includes the opossum V{lambda} sequences, shown here, reveals the interspersion of marsupial and placental mammal sequences. Although convergent evolution of metatherian and eutherian V{lambda} gene segments could account for this interspersion, the most likely explanation is the separation of the three V{lambda} lineages before the divergence of metatherians and eutherians, which probably occurred more than 100 million years ago and may have been as long ago as 175 million years (14, 15).

Mammalian VH sequences do not show a similar evolutionary interspersion between marsupials and placental mammals. There are two VH families in M. domestica, and both cluster on the same branch within the mammalian group III lineage (22). VH sequences from two other marsupial species, one a complete sequence from the North American opossum Didelphis virginiana, the other a partial sequence from the Australian brushtail possum Trichosurus vulpecula, also cluster with the M. domestica VH sequences on a common marsupial branch (Ref. 22 and our unpublished observations). Opossum V{lambda} gene segments, in contrast, retained a wider germline diversity, perhaps to compensate for less diversity in the heavy chain.

Evolution of mammalian C{lambda}

The gene duplication event that separated the mouse JC{lambda}1-JC{lambda}3 pair from the JC{lambda}2-JC{lambda}4 pair was reported to be very old, on the order of 240 million years ago, based on nonsynonomous substitution rates (40). Our analysis of mammalian C{lambda} also supports gene duplications in mice that are more ancient than those found in most mammals, as indicated by the long branch lengths for mouse C{lambda} in Fig. 7GoB. However, these duplications, like those in all mammals, not only occurred after the separation of metatherians and eutherians, probably much less than 200 million years ago, but occurred after the separation of the species themselves. The mammalian Ig{lambda} and Ig{kappa} loci have followed distinct patterns of evolution in their gene organization. The Ig{lambda} loci, in general, contain duplicated J-C units, whereas the Ig{kappa} loci have a single C segment downstream from duplicated J segments (7, 8, 9). This pattern of multiple tandem J-C duplications in the {lambda} locus in placental mammals is clearly conserved in the opossum and, therefore, conserved across mammalian orders that may be separated by as many as 175 million years. In contrast, the avian Ig{lambda}, represented by chickens and ducks, contains only a single J{lambda} and C{lambda} region (37, 38, 39), and the {lambda}-like genes in cartilaginous and boney fishes are organized in duplicated units of [VL-JL-CL] (33, 34, 35). A light chain related to mammalian {lambda} has been identified in an amphibian and found to contain more than one of each JL and CL segment, although the organization of these genes has not been reported (23). It is interesting that while the tendency to undergo J-C duplications in {lambda} is conserved across mammalian orders, the duplications themselves appear species specific and not conserved. In other words, the mammalian {lambda} locus appears to consistently evolve by duplicating the J and C segments as a unit, although the duplications present in modern mammals likely occurred after the separation of the species. The presence of paralogous J{lambda}-C{lambda} pairs within a species without orthologous relationships between species was reported by Hayzer (26) in a more extensive analyis of eutherian C{lambda} sequences. It is curious as to why the {lambda} locus in mammals would continue to independently evolve as (V)n-(J-C)n, while parallel evolution in the {kappa} locus proceeded as (V)n-(J)n-C. We have recently identified variable and constant region sequences from the opossum that are clearly the homologues of Ig{kappa} (G.H.R. and R.D.M., unpublished observations), but the complexity and organization of {kappa} in the opossum remains to be determined. It will be interesting in the future to compare how the {kappa} locus has evolved in metatherians as well.

Structure of the opossum Ig{lambda} locus and the {lambda} repertoire

The preferential use of one light chain isotype over another, as seen in many mammals, appears to correlate with the overall complexity, or number, of available VL segments, although sheep and horses may indicate that there are exceptions (3, 4). Humans have similar numbers of available V{lambda} and V{kappa} segments and use both light chain types nearly equally (60:40, {kappa}:{lambda}). Mice have a strong bias for Ig{kappa} and nearly 50-fold more V segments in their Ig{kappa} locus than Ig{lambda} (7, 9). Conversely, sheep have 10-fold more V segments in their Ig{lambda} locus than Ig{kappa}, and a 20:1 bias for Ig{lambda} expression (1, 6). The opossum, M. domestica, has ~30 V{lambda} segments that are divided among 3 evolutionarily diverse families. We would expect, although we have not yet shown, that {lambda} should contribute significantly to the expressed Ig diversity in this marsupial. When the expressed V{lambda} repertoire was sampled, V{lambda} segments from the V{lambda}1 family far outnumbered the other 2 families in V-J rearrangements cloned. Although we cannot rule out the possibility that this may reflect some bias in V-J recombination or selection for B cells expressing V{lambda}1, it is consistent with and easily explained by the number of V{lambda} segments in each family. Based on Southern blot analysis, V{lambda}1 appears to have twice as many segments as V{lambda}2 and five times as many as V{lambda}3. While it remains to be determined what percentage of the germline V segments in each family are functional, the frequency at which a V{lambda} segment is expressed likely reflects its representation in the genome, rather than a bias or preferential use.

A curious aspect of the structure of the opossum V{lambda} domains is the coincident length variation of all three CDRs. Members of the V{lambda}2 and V{lambda}3 families encode shorter CDR1 and CDR2 regions, or conversely, V{lambda}1 members encode longer CDR1 and CDR2. The V-J rearrangements generated using V{lambda}2 and V{lambda}3 segments contain shorter CDR3 regions as well. V{lambda} family-specific CDR length is also apparent in the alignment of human V{lambda} (29). Three of the human V{lambda} families (V{lambda}4, -5, and -9) have significantly longer CDR2 regions. In the opossum, the longer CDR3 found in rearrangements using V{lambda}1 does not correlate with FR4 sequence. Furthermore, in the seven rearrangements that contain a V{lambda}2 or V{lambda}3 isolated so far, four of the six putative J segments are present. These results support a lack of bias in the V{lambda}-J{lambda} recombinations and suggest that it is not the choice of J segment that creates the length variation of the CDR3 depending on whether V{lambda}1 vs V{lambda}2 or V{lambda}3 are being rearranged. The alternative explanation is that the length of the regions in the germline V{lambda}2 or V{lambda}3, which contribute to the CDR3, are shorter. We are presently cloning the germline V{lambda} segments to see if V{lambda}2 and V{lambda}3 members contain a shorter CDR3. It is also possible that during V-J recombinations involving a V{lambda}2 or V{lambda}3 there is additional nucleotide trimming at the junction to create shorter CDR3s or, conversely, more N region additions made by terminal deoxynucleotidyl transferase when a V{lambda}1 member is recombined. Shorter CDRs translate into shorter Ag binding loops in those Abs that contain a V{lambda}2 or V{lambda}3. Lack of N region additions and shorter CDR3 regions have been shown to increase Ag receptor cross-reactivity or Ag promiscuity in Ig and TCR (41, 42). It is possible that in the opossum Ig repertoire the Abs that contain a V{lambda}2 or V{lambda}3 have a broader specificity, although this remains to be experimentally determined.

PCR amplification with primers specific for opossum J{lambda} and C{lambda} segments from genomic DNA produced six unique J-C introns. Attempts to produce completely inbred lines of M. domestica have been unsuccessful to date, and we cannot presently determine whether or not the opossums we are using are homozygous or heterozygous at the Ig{lambda} locus (43). Therefore, given that we were able to clone at least six unique J-C introns, there may be as few as three functional Ig{lambda}(J-C) pairs in the opossum genome. Nonetheless, this provides the opossum with multiple functional J{lambda} segments to use in V-J recombination. Interestingly, in cattle, although multiple J-C pairs exist, the {lambda} light chain repertoire appears to be dominated by a single V-J recombination (5). As pointed out earlier, there was no apparent bias in the V{lambda}-J{lambda} recombinations in the opossum spleen cDNA library.

In summary, the {lambda} repertoire of the opossum is more heterogeneous than that of many placental mammals, such as the artiodactyls, and contains more available germline segments than rodents. The kinds of gene duplications that have occurred at the {lambda} locus in placental mammals have also occurred independently in the marsupial lineage. Given the apparent combination of any V with any J, we would predict that the overall organization of the Ig{lambda} locus is probably similar to that found in humans with an array of V segments upstream of an array of J-C pairs.


   Footnotes
 
1 This work was supported by a National Science Foundation CAREER Award (MCB-9600875) to R.D.M., and a Research Experience for Undergraduates (National Science Foundation) supplement. J.E.L. was supported by a fellowship from the Howard Hughes Medical Institute Undergraduate Research Program. Back

2 All sequences reported have been deposited in the GenBank/EMBL database and assigned accession numbers AF049746–AF049790. Back

3 Address correspondence and reprint requests to Dr. R. D. Miller, Department of Biology, University of New Mexico, Albuquerque, NM 87131-0001. Back

4 Abbreviations used in this paper: IgH, Ig heavy chain; IgL, Ig light chain; FR, framework region; CDR, complementarity determining region. Back

Received for publication July 6, 1998. Accepted for publication August 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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