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Proc Natl Acad Sci U S A. 2001 February 13; 98(4): 1775–1780. | PMCID: PMC29333 |
Copyright © 2001, The National Academy of Sciences Immunology A shark antibody heavy chain encoded by a nonsomatically
rearranged VDJ is preferentially expressed in early development and
is convergent with mammalian IgG Lynn L. Rumfelt,*† David Avila,‡ Marilyn Diaz,§ Simona Bartl,¶ E. Churchill McKinney,* and Martin F. Flajnik*†‖ *Department of Microbiology and Immunology, University of Miami School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101; †Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, MD 21201; ‡Basel Institute for Immunology, Basel 4005, Switzerland; §The Scripps Research Institute, La Jolla, CA 92037; and ¶Moss Landing Marine Labs, Moss Landing, CA 95039 Received August 3, 2000. In most vertebrate embryos and neonates studied to date unique
antigen receptors (antibodies and T cell receptors) are expressed that
possess a limited immune repertoire. We have isolated a subclass of
IgM, IgM1gj, from the nurse shark Ginglymostoma
cirratum that is preferentially expressed in neonates. The
variable (V) region gene encoding the heavy (H) chain underwent V-D-J
rearrangement in germ cells (“germline-joined”). Such H chain V
genes were discovered over 10 years ago in sharks but until now were
not shown to be expressed at appreciable levels; we find expression of
H1gj in primary and secondary lymphoid tissues early in
life, but in adults only in primary lymphoid tissue, which is
identified in this work as the epigonal organ. H1gj chain
associates covalently with light (L) chains and is most similar in
sequence to IgM H chains, but like mammalian IgG has three rather than
the four IgM constant domains; deletion of the ancestral IgM C2 domain
thus defines both IgG and IgM1gj. Because sharks are the
members of the oldest vertebrate class known to possess antibodies,
unique or specialized antibodies expressed early in ontogeny in sharks
and other vertebrates were likely present at the inception of the
adaptive immune system. In mammals, expressed Ig and
T cell receptors (TCR) of fetuses and newborns are qualitatively
different from those of adults. For Ig, a few particular variable (V)
regions are expressed by an early subset of developing B cells (B1
cells) that are later supplanted by new precursors with a diverse
repertoire ( 1). T cell precursors bearing γ/δ TCR with highly
restricted repertoires develop in the fetal thymus and then are also
replaced in this primary lymphoid tissue by conventional, major
histocompatibility complex (MHC)-restricted α/β T cells
( 2). Lymphocytes bearing such “innate” receptors are produced
early in ontogeny when little diversity is generated as a consequence
of gene (V-D-J or V-J) rearrangement ( 3– 5). In addition, innate cells
can self-renew and are found in the peritoneal cavity (B1 cells in
mice; ref. 6) and epithelial surfaces (γ/δ T cells; ref. 7),
where they are presumed to serve specialized functions. The early
appearance of innate lymphocytes, serving perhaps as a first line of
defense, and the late appearance of “adaptive” B and T cells with
great antigen receptor diversity and residing in secondary lymphoid
tissues are the foundation of the “Layering Hypothesis” of the
immune system proposed by the Herzenbergs ( 8). Elasmobranchs (sharks, skate, and rays) are members of the oldest
vertebrate taxon known to possess an adaptive immune system grounded on
Ig, TCR, and the MHC ( 9). There are at least three Ig classes in
elasmobranchs: IgM ( 10), IgNAR ( 11), and IgW ( 12– 15). IgM is the
orthologue of the IgM identified in all other vertebrates and is the
best studied class in elasmobranchs; it is found in both monomeric (7S)
and multimeric (19S) forms and comprises about half of the serum
protein in adults ( 16). Neonatal nurse sharks ( Ginglymostoma
cirratum) were shown to have very low serum levels of IgM at birth
and required over 1 month post partum to approach adult-like
levels ( 17). Their total serum protein, however, was the same as in
adults, indicating that other unidentified neonatal proteins were
present. Nurse sharks are ovoviviparous, developing within an egg case
in the uterus, so there is no maternal Ig transfer as in mammals
although there might be transfer to embryos from the yolk ( 18). All elasmobranch Ig heavy (H) and light (L) chain genes are in the
“cluster” configuration, with each H chain cluster containing one
variable (V), 2–3 diversity (D), one joining (J), and several constant
(C) exons ( 19). There are estimated to be up to 200 IgM H chain
clusters in the horn shark ( Heterodontus francisci; refs. 20
and 21). Most expressed H chains are encoded by clusters that rearrange
the V, D, and J genes in what is inferred to be the typical mammalian
fashion; in contrast, up to 50% of the clusters have V, D, and J genes
that are either partially or wholly pre-rearranged
(“germline-joined”), and these genes are not expressed at
appreciable levels, i.e., detected by Northern blotting or even as
cDNAs from adult spleen libraries ( 22). However, such germline-joined
clusters were theorized to be expressed early in ontogeny and/or in
specialized tissues; it was also suggested that they could provide a
selective advantage by perpetuating germline specificities and thus be
used for specific functions ( 22– 26). Along with Ig, TCR, and MHC, cartilaginous fish are the oldest
vertebrates to have both primary (hematopoietic cell-producing) and
secondary (immune response-generating) lymphoid tissues ( 27, 28). Based
upon morphological studies, the putative bone marrow equivalent is the
epigonal organ, which is physically associated with gonadal tissue
throughout life, or the Leydig organ, attached to the outer wall of the
esophagus. Depending on the particular species examined, either one or
both of these tissues is present and they have the same basic
organization: >50% of the cells are myeloid but lymphoid and
Ig-secretory cells have also been observed ( 29). The spleen is the only
elasmobranch secondary lymphoid tissue with defined accumulations of
mature lymphoid cells; it has distinct white and red pulp
regions but no marginal zone separating the two. The white pulp areas
contain predominantly lymphocytes of different sizes and a few mature
and developing secretory B cells. The red pulp contains erythrocytes,
leukocytes of many types (including secretory B cells), and venous
sinuses (ref. 27; L.L.R. and M.F.F., unpublished results). While carrying out studies of the developmental regulation of IgM and
IgNAR expression, we identified serendipitously a subclass of IgM
expressed during the early life of nurse sharks. As reported here, its
expression and gene structure conform to speculations tendered above
for the function of cartilaginous fish germline-joined Ig genes, and
reveal that antigen receptors expressed early in development are
different from those in adult life in a wide range of vertebrates.
Furthermore, its expression led us to reexamine, with molecular means,
the hypothesis that the epigonal organ serves as a primary lymphoid
tissue in elasmobranchs. Animals. Nurse shark ( G. cirratum) pups were delivered by Caesarian
section from three females as described ( 30). Because there is no table
of nurse shark development, we do not know the precise ages of these
animals, but offspring were collected in October/November in South
Florida when females drop their pups (mating in June/July). The pups
were maintained in aerated seawater and fed seafood twice a week. Blood
was obtained ( 16) and plasma was collected and either used immediately
for biochemical studies or frozen in aliquots at −20°C. Pup Spleen and Epigonal Organ cDNA Libraries and Genomic Cosmid
Library. Two newborn nurse shark pups were anesthetized in MS-222. Tissues were
collected, and total RNA was prepared per the TRIzol RNA isolation
protocol (GIBCO/BRL). mRNA was prepared per Ambion (Austin, TX)
Poly(A) Pure kit. The cDNA libraries were constructed per Stratagene
ZAP-cDNA library kit using 5 μg mRNA pooled from each tissue. The
cosmid library was constructed commercially by using erythrocyte
genomic DNA from one adult nurse shark per Stratagene SuperCos 1 Cosmid
library. Whole blood was centrifuged, and the peripheral blood
lymphocyte buffy coat was discarded. The erythrocyte pellet was
subjected to two rounds of Ficoll gradient centrifugation to rigorously
deplete all lymphocytes. The spleen and epigonal libraries were
screened under both high and low stringency conditions as described
( 31) with the conventional IgM V H probe ( 32). Probes. Splenic or thymic cDNA or plasmid DNA containing cloned cDNA inserts
were used as templates for PCR amplification of all probes as described
( 30). Oligo(dT)-primed cDNA was made from 5 μg nurse shark total RNA
thymus and spleen by using GIBCO/BRL Superscript First Strand cDNA
Synthesis kit. The probes were the following: conventional IgM V (no.
M92851, nucleotides 1–477); IgM 1gj V and CH 1
(no. AF327520, see Fig. A for priming sites);
recombination-activating gene 1 ( RAG1) (no.
U13982, nucleotides 6–309); terminal deoxynucleotidyl transferase
(TdT) (no. AF327519) (S.B., M.D., and M.F.F., unpublished results); the
nucleotide diphosphate kinase ( NDPK) probe was a
gift from M. Kasahara ( 33). Twenty-five nanograms of probe was labeled
with 32P[dCTP] with the Random Primed Labeling
kit (Boehringer). Typical counts were 1 ×
10 6 cpm/μl. Southern and Northern Blots. Southern blots were done under low or high stringency conditions ( 34).
Five micrograms of nurse shark pup or adult genomic DNA was digested
for 1.5 days with various enzymes. For Northerns ( 31), gels were loaded
with 20 μg total RNA or 4 μg mRNA per lane. The labeled probes were
hybridized with filters for a minimum of 20 h at 42°C and then
washed under high or low stringency conditions ( 34). Monoclonal Antibodies, Immunoprecipitation, and Metabolic Labeling. Monoclonal antibodies (mAbs) specific for nurse shark IgM were made as
described ( 11, 35, 36). CB5, CB16, and LK45 recognize both the 19S and
7S forms of IgM, GA15 prefers 7S to 19S, and GA16 prefers 19S. LK14
recognizes both 19S and 7S IgM via an L chain epitope. NART1 is
specific for the IgNAR C terminus. NK11 binds a shark cell surface
protein not present in plasma and was used as a negative control
(E.C.M. and M.F.F., unpublished results). JC4 is specific for shark J
chains (V. S. Hohman, A. S. Greenberg, L.L.R., D.A., M.F.F.,
and L. A. Steiner, unpublished results). Nurse shark plasma
proteins were labeled with 125I by using the
chloramine T method ( 37). Radio-iodinated plasma (1–4 μl containing
10 6 cpm) was incubated with 100 μl neat mAb
supernatants overnight at 4°C, and the complexes were captured with
protein-G bead adsorbents ( 11). SDS/PAGE on 5% (nonreducing) or 9%
(reducing) gels was done as described ( 11, 14). Biomax MR film (Kodak)
was used for autoradiography. For metabolic labeling, nurse sharks were
euthanized, and single cell suspensions of spleen and epigonal organ
were prepared. Cells were labeled overnight with
35S Trans (radiolabeled methionine and cysteine,
ICN) as described ( 14). Supernatants containing labeled secreted Igs
were collected and immunoprecipitated with mAbs as described above for
plasma. Protein Sequencing. IgM 1gj was immunopurified on a protein-G-mAb GA15
affinity column as described ( 36), separated on a 13% SDS/PAGE gel
under reducing conditions, and electroblotted to poly(vinylidene
difluoride) membrane (Immobilon-P sq, Millipore)
for 2 h at 250 mA constant current in 10 mM CAPS
(3-cyclohexylamino-1-propanesulfonic acid), pH 11/10% methanol.
Proteins were stained with 0.1% (wt/vol) Amido Black 1oB (Bio-Rad)
in methanol/acetic acid/water (40:10:50) and then destained with
methanol/acetic acid (50:10). Protein bands at 55.7 kDa and 53 kDa
(see Fig. ) were excised with a
razor for Edman sequencing in a Biosystems 494 (Foster City, CA)
Protein Sequencer ( 11).
IgM1gj Predominates During Early Development. While examining the ontogeny of expression of known secreted nurse
shark Ig classes, we immunoprecipitated a new form of Ig,
IgM 1gj, ** from
radio-iodinated plasma proteins of neonates with a subset of our mAbs
specific for conventional IgM (e.g., lane(s) 3 in Fig.
A). The protein recognized by the crossreacting
mAbs (GA15, GA16, and LK14) had the same biochemical properties under
reducing and nonreducing conditions in each neonate examined (16
newborn pups from 3 families), by using fresh or frozen plasma. The
H 1gj chain Mr is
≈55 kDa, suggested to be smaller by one constant domain from
conventional IgM H chains, but the same size as mammalian IgG H chains
( 38). H 1gj associates covalently with IgL chains,
determined by two-dimensional gels (first dimension under nonreducing
conditions and the second under reducing conditions; see Fig. 5, which
is published as supplemental data on the PNAS web site,
www.pnas.org). The mature IgM 1gj forms dimers
and monomers under nonreducing conditions (Fig. A);
the quantity of each form varies among individuals, which seems related
to the sharks' age (not shown). Immunoprecipitations of
IgM 1gj from metabolically labeled spleen and
epigonal organ cells from 1-week-old sharks demonstrated that it is
made de novo and indeed is the major form of Ig produced by
the pups (Fig. B, and see Fig. for mRNA expression). IgM 1gj protein is expressed in adults, but
attempts to isolate it from adult plasma were unsuccessful because of
the large amounts of conventional IgM present at maturity ( 16).
However, metabolic labeling of cells from adult epigonal organ and
spleen clearly showed IgM 1gj protein produced at
appreciable levels relative to conventional IgM only in the epigonal
organ (Fig. C). Note that the two closely spaced
H 1gj bands under reducing conditions may be the
result of differential glycosylation (there being five putative
N-linked glycosylation sites, Fig.
A and B).
The H1gj V Gene Is Germline Joined. IgM 1gj was affinity purified with the GA15 mAb
from neonatal plasma, and H 1gj was sequenced.
Both closely spaced bands described above at ≈56 and 53 kDa (Fig. )
yielded the same N-terminal amino acid sequence, DIMLTQPEAETSIXGGXLKL.
To isolate cDNAs encoding H 1gj, two cDNA
libraries were constructed with mRNA from neonatal spleen and epigonal
tissue, respectively, and screened under high and low stringency
conditions with a conventional IgM V probe. Five clones were isolated
that hybridized only under low stringency conditions and had deduced
N-terminal sequences that strictly matched the
H 1gj N-terminal protein sequence (Fig.
A). The V regions of the five clones were also
identical in sequence, with no replacements in the
complementarity-determining regions (CDRs), particularly in CDR3, the
site of greatest diversity because of somatic rearrangement of V-D-J
genes ( 38). Note that conventional IgM cDNAs were also obtained under
high stringency conditions in this library screen, with extensive CDR3
diversity generated via addition of nontemplated nucleotides (not
shown). These results suggested that a germline-joined gene encoded
H 1gj; furthermore, unlike other vertebrates
[frog ( 4, 5) and mouse ( 3)], TdT is expressed by neonatal sharks and
diversifies rearranging junctions with template-independent nucleotide
additions (also see Fig. and M.D. and M.F.F., unpublished results). To prove that H 1gj V region was germline joined,
we screened a nurse shark genomic cosmid library, prepared from
erythrocyte DNA of one shark from which all lymphocytes were rigorously
depleted, with the H 1gj V and
C H1 cDNA probes under high stringency conditions.
Isolated clones were sequenced and revealed the following:
( i) the leader exon is split by an intron that is found in
all IgV genes ( 39), indicating that H 1gj is
encoded by a typical V gene (not shown), and ( ii) the
V H1gj gene is germline joined with a V-D-J join
that precisely matches the sequences of the cDNA clones (Fig.
C). Note that we further confirmed these features of the
H 1gj gene by PCR amplification of the region
encoding CDR3 from erythrocyte genomic DNA of two other sharks (not
shown). In conventional IgM of the horn shark and ratfish ( Hydrolagus
collei), the rearrangement join generally includes two D segments,
designated D1 and D2; V H1gj CDR3 is short, in
part because it appears to use only one D gene (D2, deduced from horn
shark genomic sequences; ref. 22; Fig. A, C,
and D). It is possible that the dinucleotide TC found in the
V-D junction is the result of a P-nucleotide addition (ref. 39;
double-underlined in Fig. C). This mature V-D-J junction
appears to have been formed neither with extensive trimming nor
nucleotide addition (see Discussion and Fig. D
legend). The V domain framework regions (FR) 1–3 are most similar to horn shark
IgM V H [65% amino acid (aa) identity] but only
55% identical to any particular nurse shark IgM
V H. Phylogenetic analysis of V H1gj
indicates that it is a new nurse shark V family having only 70%
nucleotide identity and less than 70% aa identity with conventional
IgM V H (38 and see below). Besides the short
CDR3, the other conspicuous feature is the paucity of serines usually
found in a high percentage of CDR1 and CDR2 aa residues in conventional
IgM; there is an average of six serines in CDR1 and CDR2 from horn
shark ( 22), sandbar shark ( Carcharhinus plumbeus, ref. 40),
and nurse shark ( 32) H chain sequences (Fig. A). Features of the H1gj C Domains, Especially the Lack of
a CH2 Domain Exon in the Gene Clusters. The C H1 domain of IgM 1gj
lacks the cysteine (cys) commonly used for L chain association ( 38) and
is substituted by asparagine (bold, asterisk in Fig.
A). Because biochemical evidence showed that
H 1gj is disulfide bonded to L chains, we predict
that another cys, perhaps at the beginning of the
C H2 domain (asterisk in Fig.
A), forms this bridge. The biochemical analysis
above predicted only three C domains for H 1gj,
which was confirmed by the cDNA sequence. Homology searches suggested
that C H1-1gj correlates to IgM
C H1 (58% aa identity),
C H2-1gj to IgM C H3 (51%
identity), and C H3-1gj to IgM
C H4 (77% identity, Fig. A,
supplementary Table 1, and supplementary Fig. 6). Thus, like mammalian
IgG ( 38), the ancestral IgM C H2 is
“missing” in H 1gj. Sequencing of the
1750-bp intron between the C H1 and
C H2 domain exons of the
H 1gj genomic clone revealed no remnant of an
exon, showing that the ancestral IgM C H2 exon was
lost via deletion and not alternative splicing. IgM1gj Is a Single Copy Gene. Southern blot analysis was done to determine the number of
V H-1gj and C H1-1gj genes.
V 1gj and C H1-1gj exons
apparently hybridize to only one cluster under high stringency
conditions, indicating that there is only one member in this new family
(Fig. ); longer exposures did not reveal
other hybridizing genes, nor did the genomic library screen uncover
related H 1gj clusters. A conventional IgM
V H probe cross-hybridized to
IgM 1gj under low, but not high, stringency
conditions. As expected from studies in the horn shark ( 20, 21) and
skate ( Raja erinacea; ref. 41), the conventional IgM
V H probe hybridized to many genes under both high
and low stringency conditions; however, IgM 1gj
V H was detected with this probe only under low
stringency conditions (diamonds, Fig. ), consistent with the
nucleotide similarity analyses described above.
Newborn Expression and Maintenance in Adults. Because IgM1gj protein was shown to be secreted
from neonatal spleen and from both neonatal and adult epigonal organs,
a Northern blot analysis was done to ascertain which pup and adult
tissues expressed IgM1gj. The relative mRNA
expression of IgM1gj, IgM, RAG1, TdT, and the
positive control nucleoside diphosphate kinase (NDPK) is displayed in
Fig. . IgM1gj RNA
is produced at highest levels in neonatal spleen and epigonal organ and
much lower levels in the liver, kidney, and thymus. Expression is
sustained in the spleen for at least the first few weeks after birth
(Fig. B; unfortunately, the spleen RNA sample was degraded
in the oldest animal, but serum protein levels have apparently
decreased by this time, not shown). In adults,
IgM1gj RNA expression is found only in epigonal
organ, consistent with the previous immunoprecipitation analyses
(compare Figs. B and A). There are
higher amounts of IgM1gj protein in newborn
plasma relative to conventional IgM most likely because cells in both
the epigonal organ and spleen produce IgM1gj (the
exposure for IgM expression in the neonate was 80 h and the adult
only 17 h; for all other probes exposure times were the same). A
transmembrane form of IgM1gj has not been
identified from cDNA clones and may not exist.
RAG1 expression is continuous in the thymus and epigonal organ
throughout life (Fig. and data not shown). This molecular evidence
fits with the hypothesis, based on morphological grounds, that the
epigonal organ (and by inference the Leydig organ) functions as a
primary lymphoid tissue in elasmobranchs. We have discovered an IgM subclass in the nurse shark,
IgM1gj, which is expressed predominantly in
neonatal sharks. The mAbs crossreacting with
IgM1gj also recognize 7S and/or 19S IgM;
however, neonatal IgM levels are much lower than in adults, which
“allowed” the identification of IgM1gj. As
pups mature, large amounts of IgM are produced that dilute the
IgM1gj, so serum levels of the latter appear to
decrease. It is possible that, like innate lineages of mammalian
lymphocytes, the IgM1gj cell lineage develops at
the inception of the immune system and undergoes self-renewal in the
epigonal organ throughout life. Another, equally likely possibility is
that IgM1gj cells are continually produced from a
small number of stable precursors in the epigonal organ. Previous studies in the horn shark reported a very large number of
germline-joined IgH genes, but apparently no expression of them ( 22,
23). Ratfish germline-joined H chain clusters were also isolated, and
their expression could be detected only by a sensitive reverse
transcription-PCR procedure ( 42). One hypothesis to account for such
low-to-undetectable expression in adult spleen was that some of these
prejoined clusters encoding proteins with specific functions might be
expressed in a tissue-specific and/or developmentally controlled
manner; this hypothesis is fulfilled from our study of
IgM 1gj, at least for the restricted tissue
expression and ontogenetic appearance. We have detected a similar mode
of expression of an IgNAR partially germline-joined cluster (M.D. and
M.F.F., unpublished results) and L chain clusters (in collaboration
with E. Hsu, ref. 25), and we predict this expression of such clusters
will be the rule in all elasmobranchs when there is a mixture of joined
and unjoined genes of particular antigen receptor families. It may be
that the germline-joined clusters have a transcriptional advantage
early in development, and that some clusters have been selected for
particular roles before development of the adaptive response ( 25,
26). It must also be noted that, in certain L chain families, the
clusters are entirely germline joined in some elasmobranch species and
are expressed throughout life ( 43– 45). It is very likely that the H 1gj gene was derived
from a RAG-mediated rearrangement event in the germline, an activity
that was inferred from many previous studies (reviewed in ref. 23), and
recently proven for shark L chains ( 25). Proposed precise cleavages at
two heptamer/coding end junctions, and the potential P nucleotide
addition strongly suggest that the rearrangement was RAG mediated (Fig.
D). It is not known whether a germline-joined variable
(V-J) gene also encodes the covalently associated L chain. If
IgM 1gj indeed acts as an antibody, its function
may be to recognize a conserved epitope on a marine pathogen
common to nurse sharks. Alternatively, it may have a function that is
not immune related at all, e.g., acting to clear waste products
from the body, especially during development ( 46). IgM 1gj is similar to mammalian IgG by having the
domain homologous to IgM C H2 deleted over
evolutionary time in both molecules ( 38). Such deletions must have
occurred independently in genes ancestral to shark
IgM 1gj and to mammalian IgG C region genes; based
on sequence similarity to other Igs, the IgM 1gj V
family seems to have emerged some time near the divergence of nurse and
horn sharks 120–160 million years ago (see supplemental data). Its
smaller size may allow for easier passage into tissue spaces compared
with monomeric IgM; furthermore, like a mutant mammalian IgG that lacks
a hinge region ( 47), IgM 1gj may be unable to
interact with complement, thus limiting inflammatory responses in
developing sharks. Like human IgG 1 ( 48), the
disulfide bridge bonding H and L chains has been altered but in a way
difficult to predict, which might induce an atypical conformation of
the V H/V L pair. Much work is still needed as well to establish the epigonal organ as
the elasmobranch bone marrow equivalent, but the new evidence of RAG
and TdT expression only in this tissue and thymus throughout life is
compelling. Such expression of enzymes involved in rearrangement,
morphological studies demonstrating lymphoid aggregates ( 29), and the
fact that all species of cartilaginous fish have similar tissues
regardless of their location within the body ( 28, 29), strongly suggest
that epigonal/Leydig organ is a primary lymphoid tissue. Furthermore,
the data in Fig. show that, early in life, cells in the epigonal
predominantly express mRNAs encoding the transmembrane form of
conventional IgM and the secreted form of IgM 1gj,
whereas later in life high levels of secreted IgM and
IgM 1gj (as well as IgW and IgNAR, not shown for
RNA but protein levels of IgW shown in Fig. ) are found. Such data
suggest that, like mammalian bone marrow, the epigonal organ is both a
primary lymphoid tissue and a reservoir for secretory B cells. The discovery of Igs expressed early in development derived from
germline-joined genes fits well with Herzenberg's Layering Hypothesis
( 8). The dichotomy between antigen receptors expressed early and late
in development has now been described in mammals, frogs ( 49), and
cartilaginous fish, and this basic truth should herald a rich area of
future research ( 50). In particular, the elasmobranchs, with their
germline-joined gene clusters, may provide a wealth of information on
this topic; the fact that we and our colleagues have detected at least
one joined cluster expressed early in development for each antibody
class tested (and indeed only one for the IgM class in nurse sharks,
Fig. ) strongly suggests an important function for such gene products. We thank Louis Du Pasquier and Ellen Hsu for general discussion,
Becky Lohr for technical support, David Nemazee and Ferenc Livak for
critical reading of the manuscript, an anonymous reviewer for suggested
modifications, and Robyn Stanfield and Ian Wilson for discussion of the
IgM1gj structure. The work was supported by National
Institutes of Health Grant RR06603. | | TCR | T cell receptor | | | MHC | major histocompatibility
complex | | | CDR | complementarity-determining region |
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