Marsupial Light Chains: Complexity and
Conservation of λ in the Opossum
Monodelphis domestica
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J Immunol 1998; 161:6724-6732; ;
http://www.jimmunol.org/content/161/12/6724
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References
Julie E. Lucero, George H. Rosenberg and Robert D. Miller
Marsupial Light Chains: Complexity and Conservation of l in
the Opossum Monodelphis domestica1,2
Julie E. Lucero, George H. Rosenberg, and Robert D. Miller3
T
wo 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),
k and l, 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 Ref. 1). Mice and
rabbits, for example, use predominantly Igk, whereas horses,
sheep, and cattle use primarily Igl (1– 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 Vl and Vk diversity and use both extensively, 60%
Igk:40% Igl (2, 7–9). Mice, on the other hand, have only three
functional Vl segments but a large number of available Vk and
have a 95% Igk:5% Igl 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 l repertoire of cattle is restricted to a
recurrent Vl-Jl rearrangement, even though they appear to have
multiple functional Vl and Jl 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).
Department of Biology, University of New Mexico, Albuquerque, NM 87131
Received for publication July 6, 1998. Accepted for publication August 26, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
2
All sequences reported have been deposited in the GenBank/EMBL database and
assigned accession numbers AF049746 –AF049790.
3
Address correspondence and reprint requests to Dr. R. D. Miller, Department of
Biology, University of New Mexico, Albuquerque, NM 87131-0001.
4
Abbreviations used in this paper: IgH, Ig heavy chain; IgL, Ig light chain; FR,
framework region; CDR, complementarity determining region.
Copyright © 1998 by The American Association of Immunologists
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 Igl-containing
0022-1767/98/$02.00
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The Igl 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 Vl segment repertoire in the opossum was found to be
comprised of at least three diverse Vl families. Each of these families appears to be related to distinct Vl 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 Jl-Cl pairs in the opossum locus. The opossum Jl-Cl sequences are
phylogenetically clustered, suggesting that these gene duplications are more recent and species specific. Sequence analysis of a
large set of functional, expressed Vl-Jl recombinations is consistent with an unbiased, highly diverse l light chain repertoire in
the adult opossum. Overall, the complexity of the Igl 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. The Journal of
Immunology, 1998, 161: 6724 – 6732.
The Journal of Immunology
cDNAs and have found the presence of at least three highly divergent Vl families, the absence of bias in the Vl-Jl combinations, and evidence that the duplicated Jl-Cl pair arrangements
found in placental mammals is conserved in the opossum. It appears that the genetic complexity of the M. domestica Igl locus is
greater than that for the IgH locus, suggesting that l light chains
contribute significantly to the diversity of the Ig repertoire in this
species.
Materials and Methods
PCR amplification of Igl sequences
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, 53 Denhardt’s solution, 53 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
0.23 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
Isolation of M. domestica Igl cDNAs
To isolate clones containing opossum Igl sequences, fragments of
Vl 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 Vl segments (not shown).
The clones varied from 175 to 184 nucleotides in length and shared
from 52% to 74% nucleotide similarity to mammalian Vl, but less
than 40% similarity to any mammalian Vk sequences (not shown).
Two of the PCR-generated clones were from different Vl 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 l light chain
cDNAs containing variable and constant regions (Fig. 1).
Identification of three Vl families in M. domestica
The six full length cDNA sequences shown in Fig. 1 are grouped
by nucleotide similarity in the V region. The presence of at least
two Vl families, which have been designated opossum Vl1
(clones 2c, 3c, and 4c) and Vl2 (clones 7c, 10c, and 12c), is
apparent in the opossum Igl repertoire. The separation of these
sequences into two Vl 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 Vl families, an
oligonucleotide primer complementary to coding sequence near
the 59 end of the Cl 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 Vl or Jl sequences. The sequence of the Cl
primer was complementary to nucleotides 422– 441 in the Cl region shown in Fig. 1, which is a sequence common to all 6 Cl
regions found so far. A total of 40 unique Vl-Jl rearrangements
were amplified from the cDNA library and then, cloned and sequenced. Of these new sequences, the majority (36 total) clearly
grouped with the Vl1 family, while 2 grouped with the known
Vl2 family (sequences 46p and 62p in Fig. 2). The remaining 2
clones (sequences 18p and 25p in Fig. 2) shared 97% nucleotide
similarity to each other, but ,65% similarity to any Vl1 or Vl2
sequences, and defined a third Vl family, opossum Vl3. One
clone (51p in Fig. 2) was grouped as a Vl1 member but clearly
contains a FR3 from the Vl2 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.
An unusual feature that distinguishes the three families is the
presence of consistently shorter CDR1 and CDR2 regions in the
Vl2 and Vl3 families when compared with the Vl1 members.
The Vl2 and Vl3 segments are one codon shorter than Vl1 in
CDR1 and four and three codons shorter in CDR2, respectively. In
addition, the CDR3 regions created by the Vl-Jl junction are also
consistently shorter in those clones that contain rearrangements
involving Vl2 and Vl3 family members. The length of the CDR3
does not appear to correlate with a bias in Vl-Jl combinations.
Based on FR4 sequences, we estimate there to be at least six functional Jl segments in the M. domestica Igl locus (indicated by the
Roman numerals next to the FR4 sequences in Fig. 2). All six Jl
segments can be found in rearrangements that contain a Vl1 and
long CDR3 regions, while four of six Jl segments can be found in
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A degenerate oligonucleotide (59-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
Ref. 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 lZAPII 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 lZAPII. 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
59 region of M. domestica Cl (59-ACCATAGGCCATGACCATGG) was
paired in PCR with the T3 primer to amplify Vl 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 Jl-Cl pair arrangement, oligonucleotides for each known M. domestica Jl segment (JlI, 59-GTGTTCG
GCAGTGGGACCAG; JlII, 59-GTGTTCGGTGGTGGGACCAA; JlIII,
59-GTGTTCGGTGCTGGGACCAA; JlIV, 59-GTGTTCGGCCGTGG
GACCAG; JlV, 59-GTGTTTGGCGGTGGGACCAA; JlVI, 59-GTGT
TCGGCGGTGGGACCAG) were paired with the Cl primer described
above to amplify genomic fragments. Amplifications were performed using
PCR with 2 mM MgCl2 and a 60°C annealing temperature.
6725
6726
MARSUPIAL l LIGHT CHAIN SEQUENCES
rearrangements that contain a Vl2 or Vl3 with comparatively
shorter CDR3 regions. In summary, there appears to be no relationship between the combination of particular Jl with specific Vl
segments, and the length of the CDR3 region does not associate
with particular Jl segments.
To estimate the number of Vl 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. 3). A Vl1 probe hybridized to an average of 20 restriction fragments in the M. domestica genome (Fig. 3A). This
same blot was stripped and rehybridized with probes specific for
the Vl2 (Fig. 3B) and Vl3 (Fig. 3C) families, which revealed 8
and 4 genomic fragments, respectively.
Evidence for multiple Jl-Cl pairs in M. domestica
An alignment of the nucleotide (Fig. 2) or amino acid sequence
encoded (Fig. 4) 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. 2, the six sequences in Fig. 4
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 Cl sequences reveals identical
paired patterns of similarity; in other words, cDNA clones with
similar FR4 sequences share similar Cl sequences. The most
likely explanation for this pattern is the presence of multiple functional Jl segments, each with its own Cl downstream.
To confirm the presence of multiple Jl-Cl pairs in the opossum
genome, primers were designed to be unique for each of the six
known FR4 regions (Jl) and paired with the Cl primer for PCR
using genomic DNA as a target. PCR amplification with each FR4
primer paired with the Cl 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 Jl and Cl segments,
and each clone had a unique restriction map (Fig. 5). These results
confirm the presence of multiple functional Jl-Cl pairs in the
opossum genome. A Southern blot of M. domestica genomic DNA
probed for Cl revealed typically six to eight fragments (Fig. 6)
consistent with the estimate of at least six unique Jl segments
based on FR4 sequences and the presence of Jl-Cl pairs.
Phylogenetic analysis of the opossum Vl families and Cl
regions
Pairwise comparisons of the opossum sequences with Vl sequences from placental mammals revealed greater similarity between the opossum Vl families and Vl sequences from other species than that found between opossum Vl families. To illustrate
these relationships, a phylogenetic tree was constructed using
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FIGURE 1. Nucleotide alignment of six full-length cDNA clones, including 59 and 39 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.
The Journal of Immunology
6727
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FIGURE 2. Presence of three Vl 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. 1. 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.
opossum Vl aligned to representative Vl sequences from other
mammals. Fig. 7A shows a tree based on nucleotide alignment of
the FR regions of the 3 opossum Vl families and the 10 human Vl
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
6728
MARSUPIAL l LIGHT CHAIN SEQUENCES
FIGURE 3. Determination of the
number of Vl 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 Vl1 (clone mvl-5a, not
shown), Vl2 (subclone of clone 46p
in Fig. 2), or Vl3 (subclone of clone
25p in Fig. 2). Restriction enzymes
are shown as: B, BamHI; EI, EcoRI;
EV, EcoRV; H, HindIII; P, PstI; S,
SacI; X, XbaI.
that the gene duplication events that produced these families predate the evolutionary separation of mammals.
Phylogenetic trees constructed from nucleotide alignments of all
six Cl sequences from the cDNA clones in Fig. 1 with Cl regions
from other species revealed a strikingly different pattern of evolution at the C end of the opossum Igl compared with the V end
(Fig. 7B). In the case of Cl, the duplication events appear to have
occurred after speciation. The Cl regions of the opossum all cluster at the end of a long branch and, likewise, the duplicated Cl
FIGURE 4. Alignment of deduced amino acid sequences of the full-length M. domestica Igl cDNA clones from Fig. 1. Sequences are paired based on
similar constant region sequences.
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included in the alignments (not shown). The overall topology of
the tree, or relationship among the mammalian Vl sequences, is in
general agreement with that reported in more extensive analysis of
vertebrate VL sequences (26, 27). Mouse Vl2 was excluded from
the alignment because it is highly similar to mouse Vl1. The rabbit
and artiodactyl sequences cluster on their own branches, whereas
the 2 mouse sequences and 10 human Vl families are more dispersed around the tree. The opossum Vl families also intersperse
with the sequences from mice and humans. This result suggests
The Journal of Immunology
6729
FIGURE 5. Sequence and partial restriction map of Jl-Cl 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.
Discussion
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 l 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 l comparable to that described in humans. The first important conclusion from our results is that l has
been retained in the metatherian lineage. This is not unexpected
given that both l- and k-like sequences have been described in all
vertebrate groups, including sharks (23, 33–39).
Evolution of mammalian Vl
FIGURE 6. Southern blot analysis of the opossum Cl genes. Genomic
DNA was digested with the indicated restriction enzyme and probed with
a subcloned fragment of clone 7c in Fig. 1. Restriction sites are shown as:
B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SacI; Sp, SpeI; X, XbaI; Xh,
XhoI.
Phylogenetic analysis of Vl and Vk sequences from several vertebrates revealed the presence of multiple Vl groups, but only a
single Vk cluster, hence Vl has been referred to as being
“polyphyletic” compared with the Vk (26, 27). In addition, phylogenetic analysis of Vl sequences by Hayzer (26), Sitnikova and
Su (27), and Zezza et al. (36) all generally agree that human and
mouse Vl families intersperse with genes from other vertebrates,
while sequences from other species generally remain clustered
with their phylogenetic origin (i.e., all rabbit Vl clustered within
a single group, all avian Vl clustered within a single group, etc.).
Reconstruction of a phylogenetic tree that includes the opossum
Vl sequences, shown here, reveals the interspersion of marsupial
and placental mammal sequences. Although convergent evolution
of metatherian and eutherian Vl gene segments could account for
this interspersion, the most likely explanation is the separation of
the three Vl 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 Vl gene segments, in contrast, retained a wider
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regions of mouse, human, and rabbit all cluster on their own
branches. Two avian Cl sequences were included for comparison.
Mouse Cl4 was excluded because it is a pseudogene and highly
similar to Cl1. Several relationships support the validity of this
tree, including the intraspecies clustering of Cl sequences previously noted (see Ref. 26), and the common branch that the mouse
surrogate light chain Cl5 shares with Cl1; Cl5 is thought to be
derived from Cl1 (28).
6730
MARSUPIAL l LIGHT CHAIN SEQUENCES
germline diversity, perhaps to compensate for less diversity in the
heavy chain.
Evolution of mammalian Cl
FIGURE 7. Phylogenetic analysis of opossum Vl and Cl sequences. A,
Vl tree based on a nucleotide alignment of the FR sequences. Representatives of each of the 10 human Vl families (VL1–10 on the tree) were
taken from the VBASE database (29). The opossum Vl1, Vl2, and Vl3
representatives are sequences 2c, 7c, and 18p, respectively, from Fig. 2.
Sequences from the other taxa were downloaded from the GenBank database: mouse Vl1 (J00590), Vlx (D38129); rabbit Vl2 (M27840), Vl3
(M27841); cattle Vl1a (U31106); sheep Vl5.1 (M60441), Vl5.2
(AF040918). B, Cl tree based on nucleotide alignments from the opossum
sequences from the six cDNA clones shown in Fig. 1. Sequences of other
mammalian taxa and two avian species were downloaded from GenBank:
chicken Cl (K00678); duck Cl (X82069); mouse Cl1 (J00587), Cl2
(J00595), Cl3 (J00585), Cl5 (M35582); human Cl1 (X51755), Cl2
(J00253), Cl3 (J00254), Cl6 (J03011), Cl7 (M61771); rabbit Cl1
(M12388), Cl2 (M12761), Cl4 (M12763). Mouse Vl2 is very similar to
mouse Vl1 and was not included in the alignment. Mouse Cl4 is a pseudogene and very similar to mouse Cl1 and was not included in the alignment. Scale bars indicate frequency of substitutions per site.
Structure of the opossum Igl locus and the l 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 Vl and Vk segments and use both
light chain types nearly equally (60:40, k:l). Mice have a strong
bias for Igk and nearly 50-fold more V segments in their Igk locus
than Igl (7, 9). Conversely, sheep have 10-fold more V segments
in their Igl locus than Igk, and a 20:1 bias for Igl expression (1,
6). The opossum, M. domestica, has ;30 Vl segments that are
divided among 3 evolutionarily diverse families. We would expect, although we have not yet shown, that l should contribute
significantly to the expressed Ig diversity in this marsupial. When
the expressed Vl repertoire was sampled, Vl segments from the
Vl1 family far outnumbered the other 2 families in V-J rearrangements cloned. Although we cannot rule out the possibility that this
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The gene duplication event that separated the mouse JCl1-JCl3
pair from the JCl2-JCl4 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 Cl 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
Cl in Fig. 7B. 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 Igl and Igk loci have followed distinct patterns of evolution in their gene organization. The Igl loci, in general, contain
duplicated J-C units, whereas the Igk loci have a single C segment
downstream from duplicated J segments (7–9). This pattern of
multiple tandem J-C duplications in the l 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 Igl, represented by chickens
and ducks, contains only a single Jl and Cl region (37–39), and
the l-like genes in cartilaginous and boney fishes are organized in
duplicated units of [VL-JL-CL] (33–35). A light chain related to
mammalian l 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 l is
conserved across mammalian orders, the duplications themselves
appear species specific and not conserved. In other words, the
mammalian l 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 Jl-Cl pairs within a species
without orthologous relationships between species was reported by
Hayzer (26) in a more extensive analyis of eutherian Cl sequences. It is curious as to why the l locus in mammals would
continue to independently evolve as (V)n-(J-C)n, while parallel
evolution in the k 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 Igk (G.H.R. and
R.D.M., unpublished observations), but the complexity and organization of k in the opossum remains to be determined. It will be
interesting in the future to compare how the k locus has evolved in
metatherians as well.
The Journal of Immunology
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may reflect some bias in V-J recombination or selection for B cells
expressing Vl1, it is consistent with and easily explained by the
number of Vl segments in each family. Based on Southern blot
analysis, Vl1 appears to have twice as many segments as Vl2 and
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percentage of the germline V segments in each family are functional, the frequency at which a Vl 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 Vl domains is
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Vl2 and Vl3 families encode shorter CDR1 and CDR2 regions, or
conversely, Vl1 members encode longer CDR1 and CDR2. The
V-J rearrangements generated using Vl2 and Vl3 segments contain shorter CDR3 regions as well. Vl family-specific CDR length
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isolated so far, four of the six putative J segments are present.
These results support a lack of bias in the Vl-Jl recombinations
and suggest that it is not the choice of J segment that creates the
length variation of the CDR3 depending on whether Vl1 vs Vl2
or Vl3 are being rearranged. The alternative explanation is that the
length of the regions in the germline Vl2 or Vl3, which contribute
to the CDR3, are shorter. We are presently cloning the germline
Vl segments to see if Vl2 and Vl3 members contain a shorter
CDR3. It is also possible that during V-J recombinations involving
a Vl2 or Vl3 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 Vl1
member is recombined. Shorter CDRs translate into shorter Ag
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region additions and shorter CDR3 regions have been shown to
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TCR (41, 42). It is possible that in the opossum Ig repertoire the
Abs that contain a Vl2 or Vl3 have a broader specificity, although
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PCR amplification with primers specific for opossum Jl and Cl
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 Igl 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 Igl(J-C) pairs in the opossum genome. Nonetheless, this provides the opossum with multiple functional Jl segments to use in V-J recombination. Interestingly, in cattle, although multiple J-C pairs exist, the l 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 Vl-Jl recombinations in
the opossum spleen cDNA library.
In summary, the l 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 l 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 Igl locus
is probably similar to that found in humans with an array of V
segments upstream of an array of J-C pairs.
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