Marsupial Light Chains: Complexity and Conservation of λ in the Opossum Monodelphis domestica
This information is current as Julie E. Lucero, George H. Rosenberg and Robert D. Miller of September 29, 2021. J Immunol 1998; 161:6724-6732; ; http://www.jimmunol.org/content/161/12/6724 Downloaded from References This article cites 35 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/161/12/6724.full#ref-list-1
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Marsupial Light Chains: Complexity and Conservation of in the Opossum Monodelphis domestica1,2
Julie E. Lucero, George H. Rosenberg, and Robert D. Miller3
The Ig 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 segment repertoire in the opossum was found to be comprised of at least three diverse V families. Each of these families appears to be related to distinct V 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-C pairs in the opossum locus. The opossum J-C 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-J recombinations is consistent with an unbiased, highly diverse light chain repertoire in Downloaded from the adult opossum. Overall, the complexity of the Ig 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.
wo identical heavy chains paired with two identical light Our knowledge of the structure, diversity, and evolution of the
chains is the generic structure of vertebrate Ig molecules. mammalian IgL genes is based on studies of only one of the three http://www.jimmunol.org/ T In mammals, the Ig heavy chains (IgH)4 are encoded at a major orders of mammals, the eutherians or “placental” mammals. single site in the genome, but the two types of Ig light chains (IgL), To date there has been no reported IgL gene structure from either and , are encoded at separate, unlinked loci. The use of the two of the other two mammalian orders, the prototherians (egg laying IgL types can vary between species, some having a bias or pref- monotremes, e.g., the platypus) or the metatherians (marsupials). erential use of one over the other (reviewed in Ref. 1). Mice and The relationship of these three mammalian lineages has been a rabbits, for example, use predominantly Ig, whereas horses, subject of continued debate over much of this century with most sheep, and cattle use primarily Ig (1–6). The use of one IgL type investigators placing the metatherians and the eutherians together over another correlates, in general, with the overall complexity of as sister taxa, with the prototherians diverging earliest (12). How- the loci in most species. Humans, for example, have a significant ever, more recent analysis of mitochondrial DNA supports the idea by guest on September 29, 2021 amount of V and V diversity and use both extensively, 60% that prototherians and metatherians are sister taxa, with the euth- Ig:40% Ig (2, 7–9). Mice, on the other hand, have only three erians splitting off first (12, 13). Possible times for the divergence functional V segments but a large number of available V and of these groups range from less than 120 million years ago, during have a 95% Ig:5% Ig ratio (7, 9). The contributions that IgL the Cretaceous Period, to possibly greater than 170 million years make to Ab diversity can also vary greatly between species. Hu- ago, during the Jurassic Period (14, 15). A more extensive analysis mans appear to have a significant amount of light chain diversity of metatherian and prototherian immunobiology provides a com- (7, 10). In contrast, the repertoire of cattle is restricted to a parison between very distantly related mammalian species and recurrent V-J rearrangement, even though they appear to have should yield important knowledge into the evolution of mamma- multiple functional V and J segments in their germline (5). Per- lian immune systems. haps the most extreme case of limited contribution by light chains In addition to their importance to mammalian evolution, mar- occurs in the camelids (camels and llamas), which produce a form supials also provide an opportunity to study mammals that are born of IgG lacking light chains entirely (11). comparatively less developed than mice or humans. Developmen- tal immaturity combined with the lack of a placenta, which sup- ports the transfer of maternal Ig, in most marsupial species creates unique immunological problems for metatherians (16). The opos- Department of Biology, University of New Mexico, Albuquerque, NM 87131 sum, Monodelphis domestica, has been established over the last Received for publication July 6, 1998. Accepted for publication August 26, 1998. decade as an important laboratory-bred marsupial for studies of The costs of publication of this article were defrayed in part by the payment of page many areas of comparative and biomedical research (17, 18). M. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. domestica are native to South America and are a member of the family Didelphidae, which contains the largest number of species 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 within the marsupials, and Monodelphis is the most species-rich Science Foundation) supplement. J.E.L. was supported by a fellowship from the genus of the family (19). The Didelphidae are also thought to have Howard Hughes Medical Institute Undergraduate Research Program. diverged earliest from the rest of the metatheria and may contain 2 All sequences reported have been deposited in the GenBank/EMBL database and some of the oldest extant mammalian species (20, 21). assigned accession numbers AF049746–AF049790. We have begun characterizing the Ig genes of M. domestica, and 3 Address correspondence and reprint requests to Dr. R. D. Miller, Department of Biology, University of New Mexico, Albuquerque, NM 87131-0001. we previously reported that the IgH repertoire was derived from 4 Abbreviations used in this paper: IgH, Ig heavy chain; IgL, Ig light chain; FR, two related group III type VH families (22). To extend this analysis framework region; CDR, complementarity determining region. to opossum IgL, we have cloned and characterized Ig-containing
Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 6725
cDNAs and have found the presence of at least three highly di- Results vergent V families, the absence of bias in the V-J combina- Isolation of M. domestica Ig cDNAs tions, and evidence that the duplicated J-C pair arrangements To isolate clones containing opossum Ig sequences, fragments of found in placental mammals is conserved in the opossum. It ap- V segments were first amplified from a spleen cDNA by an- pears that the genetic complexity of the M. domestica Ig locus is chored PCR using a FR2-specific degenerate primer and then, greater than that for the IgH locus, suggesting that light chains cloned and sequenced. Four unique clones were found to be ho- contribute significantly to the diversity of the Ig repertoire in this mologous to the leader, FR1, and complementarity determining species. region 1 (CDR1) of known mammalian V segments (not shown). The clones varied from 175 to 184 nucleotides in length and shared from 52% to 74% nucleotide similarity to mammalian V, but less Materials and Methods than 40% similarity to any mammalian V sequences (not shown). PCR amplification of Ig sequences Two of the PCR-generated clones were from different V families, A degenerate oligonucleotide (5Ј-CCNGGYTTYTGYTGRTACCA) com- based on less than 50% similarity, and were used independently to plementary to the coding strand for the amino acid sequence WYQQKPG screen a cDNA phage library constructed from M. domestica conserved in framework region 2 (FR2) of light chain V segments (also see spleen RNA. Three clones were identified using each probe, and Ref. 23) was used to amplify opossum V fragments by anchored PCR. L The target for PCR was a commercially available M. domestica spleen all six clones were found to contain full length light chain cDNA library constructed using the ZAPII cloning vector (Stratagene, La cDNAs containing variable and constant regions (Fig. 1). Jolla, CA). The degenerate FR2 oligonucleotide was used in PCR as a Downloaded from reverse primer in combination with the T3 universal sequencing primer Identification of three V families in M. domestica specific for a site flanking the cloning site in ZAPII. Successful amplifi- cation was achieved using 2 mM MgCl2 and 55°C annealing temperature The six full length cDNA sequences shown in Fig. 1 are grouped and Taq polymerase (Perkin-Elmer, Foster City, CA). For this study, all by nucleotide similarity in the V region. The presence of at least PCR products were cloned for sequencing or for use as probes using the pCR2.1 vector (Invitrogen, Carlsbad, CA) following the manufacturer’s two V families, which have been designated opossum V 1 recommended protocol. An oligonucleotide primer complementary to the (clones 2c, 3c, and 4c) and V2 (clones 7c, 10c, and 12c), is
5Ј region of M. domestica C (5Ј-ACCATAGGCCATGACCATGG) was apparent in the opossum Ig repertoire. The separation of these http://www.jimmunol.org/ paired in PCR with the T3 primer to amplify V region segments in an sequences into two V families is based on a typically Ͼ87% unbiased manner. The spleen cDNA library described above was used as similarity among sequences in the same family and Ͻ56% simi- target with the conditions of 3.0 mM MgCl2 and 55°C annealing temperature. larity between the families. In experiments to confirm the germline J-C pair arrangement, oligo- To rapidly screen for the presence of additional V families, an nucleotides for each known M. domestica J segment (JI, 5Ј-GTGTTCG oligonucleotide primer complementary to coding sequence near Ј GCAGTGGGACCAG; J II, 5 -GTGTTCGGTGGTGGGACCAA; J III, the 5Ј end of the C region was paired with a primer specific for 5Ј-GTGTTCGGTGCTGGGACCAA; JIV, 5Ј-GTGTTCGGCCGTGG GACCAG; JV, 5Ј-GTGTTTGGCGGTGGGACCAA; JVI, 5Ј-GTGT the T3 promoter sequence flanking the cloning site in the phage TCGGCGGTGGGACCAG) were paired with the C primer described vector used to construct the cDNA library. This approach amplifies above to amplify genomic fragments. Amplifications were performed using V domain sequences, using the spleen cDNA library as target, by guest on September 29, 2021 PCR with 2 mM MgCl2 and a 60°C annealing temperature. without bias for V or J sequences. The sequence of the C primer was complementary to nucleotides 422–441 in the C re- Blot hybridizations gion shown in Fig. 1, which is a sequence common to all 6 C regions found so far. A total of 40 unique V-J rearrangements All genomic M. domestica DNA used were extracted from spleen tissue were amplified from the cDNA library and then, cloned and se- using standard protocols. For Southern blot analysis, genomic DNA were quenced. Of these new sequences, the majority (36 total) clearly cut with various restriction endonucleases following the manufacturer’s recommended conditions (see figure legends). Digested DNA were elec- grouped with the V 1 family, while 2 grouped with the known trophoresed through 1% agarose (FMC Bioproducts, Rockland, ME) and V2 family (sequences 46p and 62p in Fig. 2). The remaining 2 transferred to reinforced nitrocellulose for probing (Micron Separations, clones (sequences 18p and 25p in Fig. 2) shared 97% nucleotide Westborough, MA). Phage plaque lifts for cDNA library screening were similarity to each other, but Ͻ65% similarity to any V1orV2 also made using reinforced nitrocellulose. All probes used in this study were prepared as DNA inserts excised from plasmids and labeled with sequences, and defined a third V family, opossum V 3. One [32P]dCTP by the random primer method (Prime-it Kit, Stratagene). Hy- clone (51p in Fig. 2) was grouped as a V1 member but clearly bridizations were done at 42°C in 50% formamide, 5ϫ Denhardt’s solu- contains a FR3 from the V2 family. Whether this clone contains ϫ tion, 5 SSC, 50 mM NaPO4 (pH 6.5), 0.1% SDS, 5 mM EDTA, and 250 a bona fide germline V segment that may have undergone gene mg/ml sheared salmon sperm DNA. Final wash conditions were 65°C and conversion or recombination, or is an artifact of template jumping 0.2ϫ SSC. during PCR, remains to be determined. An unusual feature that distinguishes the three families is the Sequencing and analysis presence of consistently shorter CDR1 and CDR2 regions in the DNA sequencing reactions were performed using the ThermoSequenase V 2 and V 3 families when compared with the V 1 members. sequencing kit (Amersham, Arlington Heights, IL), and the reactions were The V2 and V3 segments are one codon shorter than V1in analyzed using an automated DNA sequencer (Perkin-Elmer ABI Prism CDR1 and four and three codons shorter in CDR2, respectively. In 377 DNA sequencer). All DNA sequences reported were derived by com- addition, the CDR3 regions created by the V-J junction are also pletely sequencing both strands of each clone. Sequences were analyzed consistently shorter in those clones that contain rearrangements using the Sequencher 3.0 program (Gene Codes, Ann Arbor, MI), and alignments were constructed using the CLUSTAL W program (24). All involving V 2 and V 3 family members. The length of the CDR3 phylogenetic trees shown are reconstructed from nucleotide alignments. To does not appear to correlate with a bias in V-J combinations. align the nucleotide sequences, first the amino acid translations were Based on FR4 sequences, we estimate there to be at least six func- aligned using CLUSTAL W with minor manual corrections, then nucleo- tional J segments in the M. domestica Ig locus (indicated by the tide sequences were aligned and gapped manually based on the protein alignments to retain codon positions. Based on these nucleotide align- Roman numerals next to the FR4 sequences in Fig. 2). All six J ments, trees were reconstructed using the neighbor-joining method of Sai- segments can be found in rearrangements that contain a V1 and tou and Nei (25). long CDR3 regions, while four of six J segments can be found in 6726 MARSUPIAL LIGHT CHAIN SEQUENCES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 1. Nucleotide alignment of six full-length cDNA clones, including 5Ј and 3Ј 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.
rearrangements that contain a V2orV3 with comparatively similar FR4 sequences share similar C sequences. The most shorter CDR3 regions. In summary, there appears to be no rela- likely explanation for this pattern is the presence of multiple func- tionship between the combination of particular J with specific V tional J segments, each with its own C downstream. segments, and the length of the CDR3 region does not associate To confirm the presence of multiple J-C pairs in the opossum with particular J segments. genome, primers were designed to be unique for each of the six To estimate the number of V gene segments present in the M. known FR4 regions (J) and paired with the C primer for PCR domestica genome, Southern blot hybridizations were performed using genomic DNA as a target. PCR amplification with each FR4 using representative clones from each of the three families as primer paired with the C primer yielded products ϳ1.8 kb long, probes (Fig. 3). A V1 probe hybridized to an average of 20 re- which were cloned and partially sequenced. Sequences internal to striction fragments in the M. domestica genome (Fig. 3A). This the primers confirmed that the amplified fragments contained an same blot was stripped and rehybridized with probes specific for intron with predicted splice sites flanked by J and C segments, the V2 (Fig. 3B) and V3 (Fig. 3C) families, which revealed 8 and each clone had a unique restriction map (Fig. 5). These results and 4 genomic fragments, respectively. confirm the presence of multiple functional J-C pairs in the opossum genome. A Southern blot of M. domestica genomic DNA Evidence for multiple J -C pairs in M. domestica probed for C revealed typically six to eight fragments (Fig. 6) An alignment of the nucleotide (Fig. 2) or amino acid sequence consistent with the estimate of at least six unique J segments encoded (Fig. 4) by the six full-length cDNAs revealed three dis- based on FR4 sequences and the presence of J-C pairs. tinct 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 Phylogenetic analysis of the opossum V families and C are grouped based on similar FR4 regions (amino acid positions regions 105–116) to illustrate the paired relationships. The FR4 regions of Pairwise comparisons of the opossum sequences with V se- cDNA clones 2c and 7c are nearly identical, and there is significant quences from placental mammals revealed greater similarity be- similarity in the FR4 regions of clones 3c and 12c, as well as 4c tween the opossum V families and V sequences from other spe- and 10c. Comparison of the six C sequences reveals identical cies than that found between opossum V families. To illustrate paired patterns of similarity; in other words, cDNA clones with these relationships, a phylogenetic tree was constructed using The Journal of Immunology 6727 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 2. Presence of three V 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 V aligned to representative V sequences from other families. Also included in the alignment were sequences from mammals. Fig. 7A shows a tree based on nucleotide alignment of mice, rabbits, and two artiodactyl species, cattle and sheep. There the FR regions of the 3 opossum V families and the 10 human V was no difference in the tree topology when CDR sequences were 6728 MARSUPIAL LIGHT CHAIN SEQUENCES
FIGURE 3. Determination of the number of V segments in the M. do- mestica genome by Southern blot analysis. Genomic DNA was di- gested with the indicated restriction enzyme, electrophoresed, blotted, and probed with a DNA fragment containing sequence that was repre- sentative of a V1 (clone mvl-5a, not shown), V2 (subclone of clone 46p in Fig. 2), or V3 (subclone of clone 25p in Fig. 2). Restriction enzymes are shown as: B, BamHI; EI, EcoRI; EV, EcoRV; H, HindIII; P, PstI; S, Downloaded from SacI; X, XbaI. http://www.jimmunol.org/
included in the alignments (not shown). The overall topology of that the gene duplication events that produced these families pre- the tree, or relationship among the mammalian V sequences, is in date the evolutionary separation of mammals. general agreement with that reported in more extensive analysis of Phylogenetic trees constructed from nucleotide alignments of all vertebrate VL sequences (26, 27). Mouse V 2 was excluded from six C sequences from the cDNA clones in Fig. 1 with C regions the alignment because it is highly similar to mouse V1. The rabbit from other species revealed a strikingly different pattern of evo- and artiodactyl sequences cluster on their own branches, whereas lution at the C end of the opossum Ig compared with the V end by guest on September 29, 2021 the 2 mouse sequences and 10 human V families are more dis- (Fig. 7B). In the case of C, the duplication events appear to have persed around the tree. The opossum V families also intersperse occurred after speciation. The C regions of the opossum all clus- with the sequences from mice and humans. This result suggests ter at the end of a long branch and, likewise, the duplicated C
FIGURE 4. Alignment of deduced amino acid sequences of the full-length M. domestica Ig cDNA clones from Fig. 1. Sequences are paired based on similar constant region sequences. The Journal of Immunology 6729
FIGURE 5. Sequence and partial restriction map of J-C 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.
regions of mouse, human, and rabbit all cluster on their own and humans. Toward improving our knowledge of immunogenet- branches. Two avian C sequences were included for comparison. ics in this species, and to gain insight into the evolution of mam- Downloaded from Mouse C4 was excluded because it is a pseudogene and highly malian Ag receptors, we have been characterizing the opossum similar to C1. Several relationships support the validity of this homologues of immunologically relevant genes including IgH, the tree, including the intraspecies clustering of C sequences previ- recombination activating gene-1, and terminal deoxynucleotidyl ously noted (see Ref. 26), and the common branch that the mouse transferase (22, 31, 32). We present here the first molecular char- surrogate light chain C5 shares with C1; C5 is thought to be acterization of a marsupial IgL. The light chain repertoire of the derived from C1 (28). opossum is derived from at least 3 ancient V families, which total http://www.jimmunol.org/ ϳ30 gene segments. These V segments appear to randomly re- Discussion combine with available J segments, giving a potential combinato- The gray, short-tailed opossum, M. domestica, has been an impor- rial diversity for opossum comparable to that described in hu- tant model for studies of marsupial immunobiology (30), but much mans. The first important conclusion from our results is that has work remains to develop the reagents needed to study marsupial been retained in the metatherian lineage. This is not unexpected immune systems at the level of sophistication achieved for mice given that both - and -like sequences have been described in all vertebrate groups, including sharks (23, 33–39). by guest on September 29, 2021 Evolution of mammalian V Phylogenetic analysis of V and V sequences from several ver- tebrates revealed the presence of multiple V groups, but only a single V cluster, hence V has been referred to as being “polyphyletic” compared with the V (26, 27). In addition, phy- logenetic analysis of V sequences by Hayzer (26), Sitnikova and Su (27), and Zezza et al. (36) all generally agree that human and mouse V families intersperse with genes from other vertebrates, while sequences from other species generally remain clustered with their phylogenetic origin (i.e., all rabbit V clustered within a single group, all avian V clustered within a single group, etc.). Reconstruction of a phylogenetic tree that includes the opossum V sequences, shown here, reveals the interspersion of marsupial and placental mammal sequences. Although convergent evolution of metatherian and eutherian V gene segments could account for this interspersion, the most likely explanation is the separation of the three V 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 FIGURE 6. Southern blot analysis of the opossum C genes. Genomic DNA was digested with the indicated restriction enzyme and probed with partial sequence from the Australian brushtail possum Trichosurus a subcloned fragment of clone 7c in Fig. 1. Restriction sites are shown as: vulpecula, also cluster with the M. domestica VH sequences on a B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SacI; Sp, SpeI; X, XbaI; Xh, common marsupial branch (Ref. 22 and our unpublished observa- XhoI. tions). Opossum V gene segments, in contrast, retained a wider 6730 MARSUPIAL LIGHT CHAIN SEQUENCES
germline diversity, perhaps to compensate for less diversity in the heavy chain. Evolution of mammalian C The gene duplication event that separated the mouse JC1-JC3 pair from the JC2-JC4 pair was reported to be very old, on the order of 240 million years ago, based on nonsynonomous substi- tution rates (40). Our analysis of mammalian C 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 in Fig. 7B. However, these duplications, like those in all mam- mals, 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 mam- malian Ig and Ig loci have followed distinct patterns of evolu- tion in their gene organization. The Ig loci, in general, contain duplicated J-C units, whereas the Ig loci have a single C segment downstream from duplicated J segments (7–9). This pattern of multiple tandem J-C duplications in the locus in placental mam- Downloaded from mals 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, represented by chickens and ducks, contains only a single J and C region (37–39), and the -like genes in cartilaginous and boney fishes are organized in
duplicated units of [VL-JL-CL] (33–35). A light chain related to http://www.jimmunol.org/ mammalian 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 inter- esting that while the tendency to undergo J-C duplications in is conserved across mammalian orders, the duplications themselves appear species specific and not conserved. In other words, the mammalian 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 by guest on September 29, 2021 species. The presence of paralogous J-C pairs within a species without orthologous relationships between species was reported by Hayzer (26) in a more extensive analyis of eutherian C se- quences. It is curious as to why the locus in mammals would
continue to independently evolve as (V)n-(J-C)n, while parallel evolution in the locus proceeded as (V)n-(J)n-C. We have re- cently identified variable and constant region sequences from the opossum that are clearly the homologues of Ig (G.H.R. and R.D.M., unpublished observations), but the complexity and orga- nization of in the opossum remains to be determined. It will be interesting in the future to compare how the locus has evolved in metatherians as well.
Structure of the opossum Ig locus and the repertoire FIGURE 7. Phylogenetic analysis of opossum V and C sequences. A, V tree based on a nucleotide alignment of the FR sequences. Represen- The preferential use of one light chain isotype over another, as tatives of each of the 10 human V families (VL1–10 on the tree) were seen in many mammals, appears to correlate with the overall com- taken from the VBASE database (29). The opossum V1, V2, and V3 plexity, or number, of available VL segments, although sheep and representatives are sequences 2c, 7c, and 18p, respectively, from Fig. 2. horses may indicate that there are exceptions (3, 4). Humans have Sequences from the other taxa were downloaded from the GenBank data- similar numbers of available V and V segments and use both base: mouse V1 (J00590), Vx (D38129); rabbit V2 (M27840), V3 light chain types nearly equally (60:40, :). Mice have a strong (M27841); cattle V1a (U31106); sheep V5.1 (M60441), V5.2 bias for Ig and nearly 50-fold more V segments in their Ig locus (AF040918). B,C tree based on nucleotide alignments from the opossum than Ig (7, 9). Conversely, sheep have 10-fold more V segments sequences from the six cDNA clones shown in Fig. 1. Sequences of other in their Ig locus than Ig, and a 20:1 bias for Ig expression (1, mammalian taxa and two avian species were downloaded from GenBank: 6). The opossum, M. domestica, has ϳ30 V segments that are chicken C (K00678); duck C (X82069); mouse C1 (J00587), C2 (J00595), C3 (J00585), C5 (M35582); human C1 (X51755), C2 divided among 3 evolutionarily diverse families. We would ex- (J00253), C3 (J00254), C6 (J03011), C7 (M61771); rabbit C1 pect, although we have not yet shown, that should contribute (M12388), C2 (M12761), C4 (M12763). Mouse V2 is very similar to significantly to the expressed Ig diversity in this marsupial. When mouse V1 and was not included in the alignment. Mouse C4 is a pseu- the expressed V repertoire was sampled, V segments from the dogene and very similar to mouse C1 and was not included in the align- V1 family far outnumbered the other 2 families in V-J rearrange- ment. Scale bars indicate frequency of substitutions per site. ments cloned. Although we cannot rule out the possibility that this The Journal of Immunology 6731
may reflect some bias in V-J recombination or selection for B cells References expressing V1, it is consistent with and easily explained by the 1. Butler, J. E. 1997. Immunoglobulin gene organization and the mechanism of number of V segments in each family. Based on Southern blot repertoire development. Scand. J. Immunol. 45:455. analysis, V1 appears to have twice as many segments as V2 and 2. Hood, L. E., W. R. Gray, and W. J. Dreyer. 1966. On the mechanism of antibody five times as many as V3. While it remains to be determined what synthesis: a species comparison of L-chains. Proc. Natl. Acad. Sci. USA 55:826. 3. Home, W. A., J. E. Ford, and D. M. Gibson. 1992. L chain isotype regulation in percentage of the germline V segments in each family are func- horse. I. Characterization of Ig genes. J. Immunol. 149:3927. tional, the frequency at which a V segment is expressed likely 4. Reynaud, C.-A., C. Garcia, W. R. Hein, and J.-C. Weill. 1995. Hypermutation generating sheep immunoglobulin repertoire is an antigen-independent process. reflects its representation in the genome, rather than a bias or pref- Cell 80:115. erential use. 5. Parng, C.-L., S. Hansal, R. A. Goldsby, and B. A. Osborne. 1996. Gene conver- A curious aspect of the structure of the opossum V domains is sion contributes to Ig light chain diversity in cattle. J. Immunol. 157:5478. 6. Foley, R. C., and K. J. Beh. 1992. Analysis of immunoglobulin light chain loci the coincident length variation of all three CDRs. Members of the in sheep. Anim. Genet. 23:31. V2 and V3 families encode shorter CDR1 and CDR2 regions, or 7. Kirschbaum, T., R. Jaenichen, and H. G. Zachau. 1996. The mouse immuno- conversely, V1 members encode longer CDR1 and CDR2. The globulin- locus contains about 140 variable gene segments. Eur. J. Immunol. 26:1613. V-J rearrangements generated using V 2 and V 3 segments con- 8. Zachau, H. G. 1995. The human immunoglobulin genes. In Immunoglobulin tain shorter CDR3 regions as well. V family-specific CDR length Genes, 2nd Ed. T. Honjo and F. W. Alt, eds. Academic Press, San Diego, p. 173. is also apparent in the alignment of human V (29). Three of the 9. Selsing, E., and L. E. Daitch. 1995. Immunoglobulin genes. In Immunoglobulin Genes, 2nd Ed. T. Honjo and F. W. Alt, eds. Academic Press, San Diego, p. 193. human V families (V 4, -5, and -9) have significantly longer 10. Ignatovich, O., I. M. Tomlinson, P. T. Jones, and G. Winter. 1997. The creation
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