University of South Florida, Department of Pediatrics, Children's Research Institute, 140 Seventh Avenue South, St Petersburg, FL 33701, USA
1 California Institute of Technology, Division of Biology 156-29, 1201 East California Boulevard, Pasadena, CA 91125, USA
2 Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA
3 Mote Marine Laboratory, 1600 Thompson Parkway, Sarasota, FL 34236, USA
Correspondence to: G. W. Litman
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Abstract |
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Keywords: epigonal organ, Ig, Leydig organ, Rag, real-time PCR, TCR, TdT
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Introduction |
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Despite extensive characterizations of many of these genes, relatively little is known with regard to the cell lineage-specific expression of Ig and TCR, their respective patterns of development, and the development of the immune repertoire. These issues are of particular significance in cartilaginous fish as these species lack an obvious equivalent of bone marrow and possess additional, unique lymphoid tissues, including the epigonal and Leydig organs, which have been described histologically as potential equivalents of bone marrow (16).
Among cartilaginous fish the oviparous clearnose skate, Raja eglanteria, is the best-characterized model that is presently available for developmental studies (17). Embryos can be obtained without sacrifice to the breeding stock and, despite a season-limited reproductive cycle, it is possible to obtain developmental-staged specimens. The studies described herein define the developmental stage- and tissue-specific expression patterns of a number of different genes that function in primary antigen recognition as well as the somatic rearrangement process, and relate these to the ontogenetic development of the immune (B cell) repertoire.
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Methods |
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DNA isolation, RNA isolation and Northern blot analysis
DNA was prepared by embedding purified erythrocytes in agarose blocks and extraction (in situ) with lithium dodecylsulfate (LDS) (18). DNA prepared in this manner consists only of the high-mol.-wt form; low-mol.-wt DNA and RNA are eliminated by passive diffusion during the extraction and block processing steps. RNA extraction using RNAzol B (Tel-Test, Friendswood, TX), mRNA selection and Northern blotting were carried out using standard methods. S26, a ribosomal mRNA, was used to normalize Northern blots as it demonstrates the most consistent relative expression of mRNA among multiple tissues (19). UV-cross-linked blot transfer membranes were prehybridized in Ultrahyb hybridization buffer (Ambion, Austin, TX) at 42°C for 30 min and hybridized with various probes that were labeled to uniform sp. act. employing random hexamers. Blots were washed under conditions of moderate stringency and exposed either to autoradiographic film or to a phosphor screen (20).
Ribonuclease protection assay
The constant regions from TCR , ß,
and
were subcloned into pBluescript (Stratagene, La Jolla, CA) (21). Probes and standards were constructed from templates using the MaxiScript kit (Ambion) following the manufacturer's protocol. RNA (10 µg) from thymus, spleen, rectal gland, epigonal, Leydig organ, spiral intestine and liver was used for each ribonuclease protection assay (RPA II kit; Ambion). Autoradiographic signals were converted into densitometric values. The highest signal for a specific mRNA type was taken as 100% relative abundance and related by fractional percentage to the determinations made for all other tissues.
Real-time PCR
Real-time PCR analysis was carried out using a GeneAmp 5700 sequence detection system (PE Biosystems, Foster City, CA). One microgram of total RNA from each tissue was reverse transcribed into cDNA using 5.5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor and 1.25 U/µl MultiScribe reverse transcriptase (PE Biosystems). Then 1 µl of each 100 µl reaction was subsequently used in each PCR along with SYBR Green PCR Master Mix (PE Biosystems) and 300 nM of each primer, constructed to optimize amplification for detection by SYBR Green. Controls with no template or cDNA controls with no reverse transcriptase were used to test each primer pair. Duplicate determinations made in the presence of DNase I gave identical results. Relative expression for each different transcript was determined by comparison to plasmid standards and cDNA values were normalized to expression of 28S rRNA (22), which was found to be abundant in all tissues at all developmental stages examined. Although the sensitivity of real-time PCR can extend to detect single-copy genes using SYBR Green, the application in these studies is the determination of relative abundance (as indicated), without assignment of specific quantifiable parameters. In multiple instances where repetitive determinations have been made (including temporal variation), the variance in results is statistically insignificant.
Genomic and cDNA libraries
A genomic library was constructed from R. eglanteria purified red blood cell DNA, which was embedded in agarose and extracted with LDS (see above). Embedded DNA was digested partially with MboI (18) and ligated into a DASHBamHI vector (Stratagene). The library is equivalent to a single genome and was amplified. Spleen, epigonal and Leydig organ cDNA libraries were constructed as described (23), and amplified from the RNA isolated from the same animal that was used as the DNA source for the genomic library.
IgM and IgX cDNA clones
Several different strategies were employed in order to selectively examine specific regions of Ig. In order to amplify cDNAs that would include the entire third complementarity-determining region (CDR3), primers were designed on the basis of known IgM and IgX sequences that spanned the variable (V) region of framework 2 (FR2) (5'-TTGGTCCGTCAGGTCCCCGGGCAG-3') to the first constant region (Cµ1) (5'-TTGATCCTCGCAGGTGAAGAGAAT-3') for IgM. The corresponding region of IgX was amplified from the V region of FR2 (5'-GGGTGAAACAGGTCCCCGGGAAAG-3') to Cx1 (5'-GAAGAGGTGATGTGGACTGAAGGC-3'). Approximately 8x105 p.f.u. from each cDNA library was plated onto nitrocellulose and replica lifts were screened with probes specific for the first constant regions of IgM and IgX. Positive clones were used as templates and amplified using high-fidelity PfuI polymerase, and the respective FR2- and Cµ1- or Cx1-specific primer sets. Spleen, gonad (which presumably contains undifferentiated epigonal tissue), liver (embryos only) and Leydig organ tissues from hatchling and 8-week embryos were isolated and cDNAs generated from DNase I-treated total RNA used in individual amplification reactions. IgM or IgX V region FR2 and the corresponding Cµ1- or Cx1-specific primers were used to amplify cDNA sequences containing CDR3. All PCR reactions were T/A subcloned. The combined PCR amplification/DNA sequencing error rate is estimated to be ~1/5000 (24).
Joined genomic IgM and IgX genes
Owing to the limited number of available heavy chain germline sequences in R. eglanteria, the FR2 primers that were used for the cDNA priming were paired with primers complementing the joining (J) region of IgM and IgX that were designed on the basis of available published and unpublished sequences. Genomic DNA that was embedded in agarose and extracted in LDS from the same animal that was used for cDNA library construction also was used for deriving genomic DNA for amplification of germline-joined genes. Cycling parameters (94°C for 1 min, 58°C for 45 s and 72°C for 30 s for 30 cycles, with PfuI polymerase) were optimized for short (250 bp) products, corresponding to fully joined genes. PCR products were size-selected (essentially only short products form), subcloned and sequenced as described for cDNA clones.
DNA sequencing and sequence analysis
Automated sequencing using the LI-COR system and ThermoSequenase (Amersham Pharmacia, Arlington Heights, IL) chemistry and analysis of sequence data were performed as described (20).
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Results |
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Differential expression of Ig heavy chain genes in multiple tissues
In the 8-week embryo, both IgM and IgX transcripts are considerably more abundant in the spleen compared to the other tissues; however, the relative abundance of IgX mRNA is greater than IgM in other tissues, including gonad, thymus, liver and Leydig organ (Fig. 5A). At this development stage, the relative abundance of the transmembrane form of IgM is greater than that of the secretory form of IgM in spleen and Leydig organ, but the secretory form is more abundant in the other tissues, in which lower levels of expression were observed (Fig. 5B
).
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The availability of considerably greater amounts of tissue in hatchlings and adults permits Northern blot analysis. In hatchlings, both IgM and IgX are expressed primarily in spleen, Leydig organ, liver and gonad. Both the long- and short-form transcripts of IgX, which are readily distinguished in Northern blot analyses, are evident in equivalent abundance in the spleen, Leydig organ and liver (Fig. 6A). Notably, there has been a significant shift from the transmembrane form to the secretory form of IgM in the 8-week embryo versus the hatchling, consistent with the presence of greater numbers of terminal differentiated cells at this later stage of development (Fig. 6B
).
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The expression of Rag-1 and TdT in development parallels the expression of Ig and TCR
Rag-1, which is integral to the rearrangement of segmentally organized Ig and TCR genes, is transcribed in several tissues in the 8-week embryo (Fig. 9A). At the hatchling stage, the relative abundance of Rag-1 transcripts is highest in the thymus (Fig. 9B
). In the adult there is only marginally significant expression of Rag-1 in the rectal gland, Leydig organ, spleen and PBL (Fig. 9C
). The expression of Rag-1 in other adult tissues occurs at or below a significant (<0.1 pg) cycle threshold. Taken together with the analyses of Ig and TCR expression, these results possibly reflect an initial wave of gene rearrangement in early embryonic development, followed by ongoing rearrangement largely in the thymus and finally, by diminished tissue-specific regulation of Rag-1 transcription.
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Germline-joined clusters of IgM and IgX are present in the germline of Raja
The sequences of only a limited number of prototypic IgM and IgX heavy chain gene clusters have been described previously (23,27,33 and unpublished observations). In order to relate the Ig gene repertoire to both ontogenetic and tissue-specific patterns of Ig gene expression, it first was necessary to determine a significant number of sequences of gene clusters across the informative region between FR2 and J, which contains CDR3. The minimum numbers of potentially productive versus non-productive sequences of germline-joined IgM and IgX genomic clusters is compared in Table 1. Of the germline-joined IgM sequences, 24 out of 53 distinctive clones contain either a frameshift or a stop codon between FR2 and the J region. By contrast, 16 of 21 distinctive IgX genomic germline-joined sequences contain either frameshifts or stop codons across the same region. Notably, in situ chromosomal hybridization has identified a far greater number of IgX- than IgM-containing loci (27). In interpreting both sets of findings, it is important to recognize that the actual number of clusters encoding presumed germline-joined pseudogenes likely is greater as deleterious substitutions could exist in other regions of the molecule.
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Of the 28 distinctive IgM cDNA sequences derived from embryos, six are out-of-frame. Notably, the sequence across the FR2 J region of one of these out-of-frame transcripts is identical to a corresponding sequence of an out-of-frame genomic clone, indicating that this gene can be expressed. Similarly, two of 22 in-frame cDNA sequences, both of which derive from embryonic liver, can be matched to the nucleotide sequences of two different in-frame germline-joined genomic clones. Twenty-five IgM cDNA sequences from hatchlings also were compared with the genomic sequence database. Two of the in-frame sequences from hatchling gonadal tissue are identical at the nucleotide level to two different in-frame germline-joined genomic clones. However, none of the 99 adult IgM cDNA sequences, all of which are in-frame, match the sequences in the germline-joined gene database. None of the adult IgM cDNAs that were examined match the sequences that were derived in parallel analyses of the embryonic or hatchling IgM cDNAs.
Of the IgX embryonic cDNA sequences, nine out of 10 (on the basis of the FR2 J sequence) represent productive (in-frame) transcripts; of 25 hatchling sequences, 21 were in-frame. All of the 158 total IgM and IgX cDNA sequences in the adult potentially are productive. Finally, no matches across this region were found between embryo, hatchling and adult cDNAs or between these partial cDNAs and the database of germline-joined genes.
CDR3 diversity is equivalent at different developmental stages
cDNA sequences were examined for CDR3 diversity. No significant differences in overall CDR3 diversity and length were observed between the cDNAs recovered from different tissues at the three different developmental stages that were examined (Fig. 10). Specifically, the lengths of CDR3 for IgM range from 3 to 12 residues in the embryos, 3 to 13 residues in the hatchlings and 3 to 15 residues in the adults. Similarly, the lengths for CDR3 for IgX range from 3 to 10 residues in the embryos, 3 to 11 residues in the hatchlings and 2 to 14 residues in the adults. Although several cDNAs were identified in hatchling and adult that were longer than those recovered in embryos, this difference is minimal and largely reflects individual outliers as well as the considerably larger sample size in adults.
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Discussion |
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TCR and Ig expression in ontogeny
In order to approach the analysis of gene expression in these species at periods in development in which only limited amounts of certain tissues are available, it was necessary to utilize several different complementary approaches, including real-time PCR, ribonuclease protection assays and Northern blot analysis. Taken together, these approaches have revealed both conserved and divergent patterns of expression of genes that are critical to lymphoid development and function.
During embryonic development, all four classes of TCR are expressed primarily in the thymus starting at a point two-thirds of the way through embryonic development, which is similar to the timing of the onset of TCR expression during the embryonic development in mouse (35). High levels of expression of both Rag-1 and TdT, which function in segmental rearrangement, are evident in the embryonic thymus, consistent with rearrangement and junctional diversification of TCR. In hatchlings, TCR expression is detected in the spleen and intestine, implicating these tissues as potential secondary lymphoid sites. The lack of abundant expression of TCR , ß,
and
in the thymus at the hatchling stage may relate to a `lull' in the waves of thymic precursor gene expression, such as has been documented in an avian model (36). In the adult, the coordinate expression of TCR
and ß in the thymus and spleen correspond to the patterns evident in mammals where these two tissues are the sites of primary
ß T cell development and
ß T cell function respectively.
TCR expression cannot be detected in the adult unless a higher sensitivity ribonuclease protection analysis is employed; studies using this method indicate that a reciprocal relationship exists between TCR
and
in thymus and spleen (Fig. 4B
). This observation also is interesting from the standpoint of cis regulation of gene expression (37); however, the genomic relationship of TCR
and
has not yet been defined in skate, which limits further interpretation of this finding. It remains to be seen whether the unique expression pattern of TCR
corresponds to a lymphoid or non-lymphoid function. Although it is tempting to relate the expression of TCR genes in skate to that of the orthologous forms of TCR found in mammals, it is important to recognize that the dimerization patterns of the former are not known. Furthermore, it remains possible that the specific function of these molecules may differ from those of the orthologous forms seen in higher vertebrates.
A far more complex pattern emerges for the tissue-specific expression of Ig genes during ontogeny. The highest relative abundance of both IgM and IgX also occurs at 8 weeks of embryonic development and then falls off dramatically. Significant lymphoid gene expression is seen in the embryonic but not adult liver of skate, paralleling the tissue-specific expression pattern that occurs in mammals (38,39). At 8 weeks of embryonic development, the highest relative abundance of IgM and IgX is seen in the spleen; IgX is expressed in greater abundance in more tissues relative to IgM at this stage. It presently is difficult to speculate as to the significance of this observation in that the function of IgX is not understood and the expression of both classes of light chain genes, which are germline joined, is uncoupled from the segmental rearrangement process. However, the distinctly regulated patterns of IgM and IgX expression as well as expression of light chain genes suggests that the variation observed is functionally significant, and that separate lineages of cells may express IgM and IgX at these stages.
The abrupt coincidental expression of Ig and Rag-1 genes, in the spleen, liver, Leydig organ and gonad in the 8-week embryo suggests that B cell development occurs at multiple sites in the developing skate embryo in contrast to the apparent restriction of T cell development to the thymus. Moreover, in the embryonic and hatchling skate there is substantial Ig gene expression in the thymus, raising the possibility that the thymus also could be a site of B cell development in these early stages. In other vertebrates, variation in the sites of B cell development, such as the avian bursa (40), are in marked contrast to T cell development.
Although both Rag-1 and TdT are involved in the generation of antigen recognition site diversity, the tissue-specific expression patterns of these genes differ. The overall pattern for Rag-1 expression is relatively uniform in a number of different lymphoid tissues in the embryo, and is followed by a disproportionate increase in abundance in the thymus of the hatchling and then exhibits a reduced level of expression in a number of different tissues in the adult. However, the apparent uniformity of Rag-1 expression may or may not reflect protein levels. The less restricted nature of Rag expression is potentially significant against the background of germline-joined gene clusters. Furthermore, differences in Rag expression seen in cartilaginous fish may relate to the 23 orders of magnitude reduction in intronic length separating recombining elements in cartilaginous fish and the lack of combinatorial diversity during genetic rearrangement in these species. TdT expression is elevated in thymus in the embryo, but is hardly detectable in only three lymphoid tissues at the hatchling stage and in the intestine of the adult. In these studies, there is no apparent correlation between TdT expression and the diversification of the repertoire, although it plays a highly significant role in repertoire development in mammals (41). However, distinct differences are apparent in terms of Ig repertoire diversity in skate versus mammalian ontogeny (see below).
Roles of the Leydig and epigonal organs
The highest levels of expression of IgM and IgX heavy chains as well as LCI and LCII are found in the Leydig organ, which is unique to certain species of cartilaginous fish. Even employing a highly sensitive ribonuclease protection assay, it was not possible to detect significant levels of TCR mRNAs in either the Leydig or epigonal organs, which both express high relative levels of Ig heavy and light chain mRNAs. The absence of TCR expression distinguishes the Leydig organ from other lymphoid tissues, including avian bursal tissue in which a similar inability to detect TCR mRNAs using Northern blotting has been noted; however, TCR expression in the bursa can be detected using RT-PCR. T cell seeding to bursa or contamination with PBL represents possible sources for the signals (C.-L. Chen, pers. commun.).
In terms of understanding the role of the Leydig organ in B cell development, it is notable that Rag-1 is expressed most abundantly in the adult Leydig organ but TdT expression is reduced significantly relative to the levels that are expressed in the adult intestine, which are roughly equivalent to the levels of TdT that are expressed in the thymus. The relative diversity of the CDR3 region of IgM and IgX cDNAs from Leydig organ is not significantly different from that observed in cDNAs recovered from other tissues. Inspection of the Leydig sequences reveals no unusual patterns of predicted residues (data not shown). Although the most distinctive feature of the Leydig organ is largely of a quantitative nature, it is likely that the organ plays a significant role in the adaptive immune response.
Recently we identified a new member of the PU-1 family of transcription regulatory factors and have designated it as SpiD (42). Analyses of expression patterns using real-time PCR have shown that SpiD, as well as PU.1, are expressed abundantly in the Leydig organ and epigonal but not in spleen; whereas SpiC is not expressed in Leydig organ and epigonal but is expressed abundantly in spleen. In that both T and B cells are abundant in the spleen and B cells are highly abundant in Leydig organ and epigonal, it is probable that B cells in Leydig organ and epigonal are either at a different stage of development or represent different lineages than those found in spleen, analogous to B1 versus B2 lineage (43) and other functional distinctions in repertoires of mature B cells (44).
Interestingly the epigonal, another lymphoid organ found in those species, is notably larger in cartilaginous fish that lack a Leydig organ (45) and to some degree the tissue-specific expression patterns defined here for the Leydig organ resemble those seen in the epigonal organ. Furthermore, both tissues express equivalent amounts of both light chain types in the adult, but heavy chain gene expression is much higher in Leydig organ. Although it is beyond the present scope of these studies, the results are consistent with the possibility that the epigonal and Leydig organs serve either redundant or complementary roles in lymphoid development.
Differential regulation of Ig gene cluster expression in development
The relative levels of IgM and IgX do not vary in relation to each other throughout development; however, significant variation in the expression of the two different families of germline-joined light chain genes is apparent. Specifically, the relative abundance of LCI expression is consistent during ontogeny, in marked contrast to the relatively low level of expression of LCII in the embryo and hatchling. However, LCII in the adult skate is the predominantly expressed form of light chain that is expressed in the Leydig organ. Efforts presently are underway to determine whether or not there is any specificity in utilization of the various subfamilies of joined light chain genes. Although interpretation of these data is confounded by the paucity of information regarding the association patterns of Ig heavy and light chains in cartilaginous fish, the observations described here provide critical information that will facilitate a better understanding of such interactions and the role of allelic exclusion in the regulation of Ig gene expression in cartilaginous fish.
In order to provide an initial estimate of the diversity of the immune repertoire and in particular to examine the expression of germline-joined genes, a large number of Ig cDNAs from spleen, Leydig organ and epigonal derived from a single adult animal were sequenced and their CDR3s were compared. The basis for these types of studies lies in the studies of CDR3 diversity that have been conducted in a number of different higher vertebrate species, and have established an age-dependent increase in CDR3 length and diversity (4649). Within the sample size represented, no structurally significant differences are apparent in terms of overall CDR3 length and diversity. Several in-frame cDNA sequences (FR2 Cµ1), which were identified in both the embryonic liver and hatchling epigonal organ, are indistinguishable from the corresponding regions of in-frame germline-joined genomic sequences. None of the significantly greater number of FR2
Cµ1 and FR2
Cx1 (n = 158) cDNA sequences recovered from an adult appear to have derived from germline-joined clusters based on comparison of CDR3 sequences using the database of genomic germline-joined CDR3 sequences that was generated specifically for this study. The findings indicate that transcription of germline-joined IgM and IgX clusters may constitute ~1015% of the Ig expression seen in embryo and hatchling; these genes are not expressed in the adult skate. These latter observations are consistent with past failures to detect transcripts of germline-joined heavy chain genes that have been carried out using tissues derived from adults (10). However, it also is possible that later in development transcripts of somatically rearranged genes are too abundant to permit detection of the rare potential transcripts that derive from germline-joined genes.
Developmental regulation selection for in-frame rearrangements
It is critical to note that a fair number of non-productively rearranged IgM and IgX transcripts also were observed in the embryo (>25%), as well as in appreciable numbers in hatchling tissues (see Table 1). Such transcripts contain frameshifts or stop codons in the CDR3 region and would result either in truncations or reading frame shifts in the J regions. In contrast, all of the sequences analyzed from adults were in-frame. This suggests that the adult repertoire is actively biased for successful protein expression through a mechanism that does not operate in earlier life. Clonal selection of B cells or selective mRNA stabilization by polysomes are examples of mechanisms that might contribute in a developmental-regulated way. Although it is tempting to speculate that the `joined' genes may have some function in the embryo, expression of germline-joined genes, both in-frame and out-of-frame, may reflect a generalized transcription phenomenon that occurs in early development as opposed to a cluster-specific, functionally relevant event in early development. It is clear that the burst of transcriptional activity at 8 weeks could be accompanied by transcriptional activation of dominant gene loci that are not expressed at later developmental phases (see below). Recently, similar expression of a germline-joined heavy chain gene has been observed in the epigonal organ of neonatal nurse shark, which lacks a Leydig organ, but not in the adult epigonal organ (M. Flajnik, pers. commun.).
The increase in Ig and TCR gene expression at the 8-week developmental stage correlates with the apparent surge in the expression of Rag-1 and TdT. Based on the coincidental increases in transcriptional activity and high proportion of non-productive transcripts at 8 weeks, mass transcription of antigen receptor gene clusters may be taking place. The nature of Ig organization in the skate (and other cartilaginous fish) is possibly prone to non-productive transcription as distances between promoters and the coding segments of Ig loci are orders of magnitude closer than are found in mammals (1). Widespread run-off of Ig clusters in early development may establish the B cell antigen binding receptor repertoire, which is regulated further as cells with productively rearranged Ig mature. Establishment of a repertoire early in development by such a `shotgun' approach to transcription would negate a requirement for precise regulation of selective transcription of clusters in the mature adult, which are present on different chromosomes (27). Examples of the use of strategies that differ from those used in man and mouse to establish an Ig repertoire have been defined in other classes of vertebrates, including the bursa of Fabricius in avians (40,50) and ovine Peyer's patches (51,52), as well as in the lymphoid tissues of rabbit (53) and other mammals (54,55).
General similarities in the ontogeny of T cell development are evident between observations made in this study and those established in other model systems. On the other hand, the unique clustered genomic organization and expression of Ig differ markedly in terms of both the expression patterns and involvement of unique lymphoid tissues. An explanation, at least in part, for these observations is that T cells and TCR ( and ß) may be constrained in an evolutionary sense owing to their obligatory interactions with MHC for antigen recognition, necessitating a parallel, cooperative evolution of the two (TCR and MHC) systems. In contrast, B cells may lack direct dependence on a separate multigene family, thus explaining the marked differences in organization, isotype and diversification mechanisms. Such differences may accompany the variation seen in the sites of primary lymphopoiesis that have been observed throughout the different classes jawed vertebrates (1).
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Acknowledgments |
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Abbreviations |
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CDR complementarity-determining region |
FR framework region |
LCI light chain type I |
LCII light chain type II |
LDS lithium dodecylsulfate |
PBL peripheral blood lymphocytes |
Rag recombination-activating gene |
TdT terminal deoxynucleoside transferase |
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Notes |
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Received 15 December 2000, accepted 10 January 2001.
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References |
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