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Multiple Sites of V Diversification in Cattle¹

Mark R. Lucier, Rachel E. Thompson^*, James Waire, Athena W. Lin, Barbara A. Osborne^, and Richard A. Goldsby^2,,*,

^* Department of Biology, Amherst College, Amherst, MA 01002; and Department of Veterinary Sciences and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ig repertoire diversification in cattle was studied in the ileal Peyer’s patch (IPP) follicles of young calves and in the spleens of late first-trimester bovine fetuses. To investigate follicular diversification, individual IPP follicles were isolated by microdissection; V diversity was examined by RT-PCR and subsequent cloning and sequencing. When 52 intrafollicular sequences from a 4-wk-old calf were determined and compared, two major groups, one of 23 members and the other of 25, could be delineated. An examination of these groups revealed clear genealogic relationships that implicated in situ diversification of V sequences within the confines of an IPP follicle. V expression was also examined in early (95 and 110 gestational day) fetal bovine spleens. Although earlier studies in cattle and sheep implicated the IPP as a likely site of Ab diversification, a close investigation of V sequences in late first-trimester fetal calves revealed that diversity appears in the early fetal spleen before the establishment of a diverse repertoire in the ileum. When the sequences for the fetal spleen were compared with an existing pool of germline sequences, we found evidence of possible gene conversion events and possible untemplated point mutations occurring in sequences recovered from fetal spleens. We conclude that IPP is not the sole site of V diversification in cattle. Also, as suggested for rabbits, cattle may use both gene conversion and untemplated somatic point mutation to diversify their primary V repertoire.

	Introduction

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Anumber of different mechanisms, including rearrangement, combinatorial association, gene conversion (GC)³ and somatic hypermutation, have evolved to diversify the primary Ig repertoire. Different species exemplify the use of one or more of these pathways. Humans and mice generate the bulk of diversity through the rearrangement of multiple V(D)J gene segments (1, 2). Additional diversity can be generated junctionally and through terminal deoxynucleotidyltransferase additions (3). Swine appear to rely heavily on rearrangement, and their Ig sequences are remarkably similar to humans (4). The best studied example of GC as a primary mechanism of diversification is the chicken, in which a single V(D)J or V(J) rearrangement is diversified by GC employing a donor pool of V pseudogenes located upstream of the rearranged gene (5, 6). Studies by Reynaud et al. have been interpreted to indicate that sheep use untemplated somatic point mutations (pm) to diversify light chain genes within the follicles of the ileal Peyer’s patch (IPP) (7). Other studies in the rabbit system (8) have shown that these animals use limited and preferential rearrangement (9) coupled with a combination of GC and untemplated somatic pm (10) to diversify the primary repertoire. Other systems such as shark (11, 12, 13), frog (14, 15), and teleost fish (16) provide an additional perspective on the spectrum of strategies used by vertebrates to diversify the primary repertoire.

In contrast, the mechanisms used to diversify Ig genes in cattle await clarification. Hansal (17) and Parng (18) found that the light chain repertoire of cattle is extensively diversified in the IPP of cattle by 11 days of age. By examining germline gene segments, Parng et al. (19) found that the pool of V segments contained both functional genes and nonfunctional pseudogenes. A role for GC in the diversification of V was suggested by the observation that when expressed genes of mass IPP tissue isolated from a cDNA library were sequenced and compared with germline counterparts, donor sequences from germline genes were found inserted in functional genes. However, the mRNA was obtained by extraction of a section of IPP that contained many follicles, and it was not possible to establish progenitor-progeny relationships among the sequences. Consequently, it was not possible to provide direct evidence that the conversion events actually took place within the confines of IPP follicles. We felt that the establishment of progenitor-progeny relationships among V sequences obtained from a single follicle would demonstrate that the IPP follicle is indeed a site in which V genes diversify. The rationale for this approach is that the likelihood of a collection of genealogically related clones arising elsewhere in an animal and by chance populating the same follicle are vanishingly small.

To examine the mechanisms of V diversification within the IPP, individual follicles were removed from the IPP by microdissection; the diversity of the B cell repertoire was examined within the microenvironment of an individual follicle. Intrafollicular V region sequences were then compared with each other to establish possible genealogic relationships among the sequences. Pioneering studies in mice used genealogic analysis to demonstrate somatic hypermutation in germinal centers (20). Subsequently, this method of analysis has been used to uncover patterns of mutation consistent with GC and somatic hypermutation in the chicken (21) and rabbit (10). In these studies, a consideration of intrafollicular genealogies was helpful in exploring the mechanisms used to diversify V regions within the IPP. In this work, genealogic trees assembled from light chain V sequences obtained from individual follicles indicate that both GC and untemplated somatic pm could be involved in V diversification within IPP follicles.

To identify other sites and times of V diversification in cattle, we surveyed organs and tissues of first-trimester bovine fetuses for the expression of V. Such a survey revealed that only the spleen expressed an extensively diversified V repertoire. Furthermore, a comparison of V sequences from a first-trimester fetus with a library of bovine V germline genes revealed that both GC and untemplated somatic pm were contributing to the V repertoire of the fetal spleen. These observations and the genealogic studies mentioned above led to the conclusion that the spleen is an early site of V diversity in cattle. The development of V diversity in the spleen precedes the advent of diversity in intestinal tissue. Furthermore, it was demonstrated using the genealogic approach outlined above that, in addition to being sites of V diversity, IPP follicles are actual sites of V diversification.

	Materials and Methods

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Animals

The experimental animals used in this study were obtained from either the University of Massachusetts Dairy Farm (South Deerfield, MA) or a local slaughter house. The internal organs of immunologic interest (spleen, liver, thymus, intestine, and bone marrow) were harvested and stored on ice until use. Blood was diluted into Alsever’s solution and chilled on ice.

Isolation of IPP follicles

The ileal-coecal section of the intestine was isolated, and a small section was removed. The section was cut open and quickly rinsed three times with ice-cold PBS. The section was then placed into a container of fresh PBS and kept ice-cold. Using fine forceps, scissors, and a dissection microscope, individual follicles were surgically isolated from the intestine. The isolated follicles were placed in 600-µl bullet tubes with 100 µl of Ultraspec RNA (Biotex Labs, Houston, TX).

RNA isolation

RNA was extracted from tissue or cells using the Ultraspec RNA isolation system as directed by the manufacturer. The resultant RNA pellet was dissolved in diethyl pyrocarbonate (Sigma, St. Louis, MO) -treated water and quantified by a Lambda 3B spectrophotometer (Perkin-Elmer, Norwalk, CT).

To isolate RNA from individual follicles, the manufacturer’s instructions were followed; 10 µl of sterile 3 M sodium acetate and 40 µg of glycogen per 100 µl of Ultraspec RNA were added at the isopropanol precipitation step. The isopropanol precipitation was allowed to proceed overnight at -20°C.

RT-PCR conditions

To obtain cDNA from the follicles, the RNA pellet was reverse transcribed using the Moloney murine leukemia virus reverse transcriptase kit (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions with deoxynucleoside triphosphates from Promega (Madison, WI). The resultant cDNA was amplified using the Taq polymerase kit (Life Technologies) with deoxynucleoside triphosphates from Promega as directed by the manufacturer. To amplify the V region of the light chain, a leader primer (5'-CTC-TCT-GCA-CAG-GAT-CCT-GGG-C-3') and a joining region primer (5'-CAG-GAC-GGT-CAG-TGT-GGT-CCC-GC-3') (Amitof, Boston, MA) were used. PCR conditions were as follows: initial melt at 95°C for 3 min followed by 30 cycles of a two-step program (1 min at 95°C and 2 min at 72°C). The reactions were then held at 75°C for 5 min and cooled to 4°C.

The resultant PCR products were visualized by ethidium bromide staining after running on a 2% agarose gel. The light chain primers yielded a band of 358 bp.

Cloning, plasmid extraction, and sequencing

PCR products were ligated into the pCR^TM11 vector according to the manufacturer’s instructions (Invitrogen, San Diego, CA) and transformed into competent DH5 cells. The transfected bacteria were then plated onto Luria-Bertani (LB) plates containing 40 µg/ml ampicillin (Sigma) and 40 µg/ml 5-bromo-4-chloro-3-indolyl ß-D-galactoside (Boehringer Mannheim, Indianapolis, IN). White colonies from the transformed plates were grown overnight in LB media (Life Technologies), and plasmids were isolated using a Wizard Plus Minipreps DNA Purification System (Promega) as directed by the manufacturer. The resultant plasmids were checked for inserts by PCR screening with the light chain primers. The positive plasmids were then sequenced using a USB Sequenase Kit (Amersham, Cleveland, OH) as directed by the manufacturer. All sequencing was done in both directions using ]³²P[dATP (Amersham, Arlington Heights, IL). Sequences were analyzed using the Wisconsin Package Genetics Computer Group (GCG) database program (Madison, WI).

Taq error experiment

An individual clone containing a 110-day-old fetal liver V gene was subjected to three separate PCR reactions using the leader primer (5'-CTC-TCT-GCA-CAG-GAT-CCT-GGG-C-3') and the joining region primer (5'-CAG-GAC-GGT-CAG-TGT-GGT-CCC-GC-3') (Amitof). PCR conditions were as follows: initial melt at 95°C for 3 min followed by 30 cycles of a two-step program (1 min at 95°C and 2 min at 72°C). The reactions were then held at 75°C for 5 min and cooled to 4°C. The resultant PCR products were visualized for the 358-bp light chain band by ethidium bromide staining after running on a 2% agarose gel.

Each of the PCR reactions was then ligated into the pCR^TM11 vector according to the manufacturer’s instructions (Invitrogen) and transformed into competent DH5 cells. The transfected bacteria were then plated onto LB plates containing 40 µg/ml ampicillin (Sigma) and 40 µg/ml 5-bromo-4-chloro-3-indolyl ß-D-galactoside (Boehringer Mannheim). White colonies from the transformed plates were grown overnight in LB media (Life Technologies), and plasmids were isolated using a Wizard Plus Minipreps DNA Purification System (Promega) as directed by the manufacturer. The resultant plasmids were checked for inserts by PCR screening with the light chain primers. The positive plasmids were then sequenced using a USB Sequenase Kit (Amersham) as directed by the manufacturer. All sequencing was done in both directions using [³²P]deoxyATP (Amersham). Sequences were analyzed using the Wisconsin Package GCG database program. A total of 30 colonies (10 from each of the three PCR reactions) were sequenced and analyzed.

	Results

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V diversification within a single 4-wk-old IPP follicle

The V region genes of 52 clones from a single IPP follicle of a 4-wk-old Holstein calf were sequenced and analyzed. Two major groups could be delineated within the follicle studied. We observed <85% similarity between these groups and >94% similarity within a single group. These sequences are displayed in abbreviated form in Figs. 1 and 2. Of the 52 sequences obtained from a single follicle, 23 were assigned to group A and 25 to group B. The remaining four clones sequenced from this follicle did not fit into either group.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Group A abbreviated V region sequences from an isolated IPP follicle. Only triplets with nucleotide differences are depicted. The number above each column indicates the location of the triplet in the V region. All clones were double-strand sequenced, with the exception of clone 29 (marked with an asterisk). The sequence R7 found by Parng et al. (19) is a germline gene and is the suspected founder clone for this group. Suspected GCs are indicated by uppercase letters; suspected pm are indicated by lowercase letters.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Genealogic tree for group A from an individual IPP. The above genealogic tree was derived from sequence analyses of V light chain regions. The block denotes the root sequence of the tree; in this case, the root sequence was the bovine V germline gene R7 that was found by Parng et al. (19). The numbers within the circles depict the clones that have descended from this root clone. Numbers within the ellipses depict the isolation of more than one clone of identical sequence. The notations next to the arrows indicate the number of mutations differentiating the clones and whether the mutation was scored as a templated event (GC) or an untemplated event (pm).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Group B abbreviated V region sequences from an isolated IPP follicle. Only triplets with nucleotide differences are depicted. The number above each column indicates the location of the triplet in the V region. Clone 40 is the suspected founder clone for this group. Suspected GCs are indicated by uppercase letters; suspected pm are indicated by lowercase letters.

Group B contains more diversity than observed in group A; the resultant tree (Fig. 4) reflects this diversity with a greater number of branches. We found a total of 42 base-pair changes that could be traced back to possible donors in the pool of germline genes found by Parng et al. (19). In addition to the potential conversion events, we observed 18 mutations that could not be traced to a germline donor. These are labeled as possible untemplated somatic pm.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Genealogic tree for group B from an individual IPP. The above genealogic tree was derived from sequence analyses of V light chain regions. The block denotes the root sequence of the tree. In this case, a clone was determined by PILEUP analysis to be the founder clone within this group. The numbers within the circles depict the clones that have descended from this root clone. Numbers within the ellipses depict the isolation of more than one clone of identical sequence. The notations next to the arrows indicate the number of base changes differentiating the clones and whether the mutation was scored as a putative templated event (GC) or a putative untemplated event (pm).

V diversification within a single 3-mo-old IPP follicle

A total of 40 clones from a single IPP follicle of a 3-mo-old Holstein calf were sequenced and analyzed. One major group could be delineated within this follicle. The seven sequences in group C are displayed in abbreviated form in Fig. 5. A genealogic tree was derived for group C (see Fig. 6).

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Group C abbreviated V region sequences from a 3-mo-old IPP. Triplets with nucleotide differences are depicted. The number above each column indicates the location of the triplet in the V region. Base-pair deletions are indicated with an asterisk. The sequence R18 found by Parng et al. (19) is a germline gene and is the suspected founder clone for this group. Suspected GCs are indicated by uppercase letters, and suspected pm are indicated by lowercase letters.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Genealogic tree for group C from an individual IPP. The above genealogic tree was derived from sequence analyses of V light chain regions. The block denotes the root sequence of the tree; in this case, the root sequence was the bovine V germline gene R18 that was found by Parng et al. (19). The numbers within the circles depict the clones that have descended from this root clone. Numbers within the ellipses depict the isolation of more than one clone of identical sequence. The notations next to the arrows indicate the number of base changes differentiating the clones and whether the mutation was scored as a templated event (GC) or an untemplated event (pm).

V diversification in tissues of fetal calves

Ig genes from the early fetal spleen, liver, blood, and ileum were examined for the expression of V. RNA extracted from each of these tissues was reverse transcribed and subjected to PCR using primers designed to amplify 358 bp of V, which included the framework regions and complementary determining regions. It was found that V was expressed in the fetal spleen, liver, ileum, and blood (see Fig. 7). The amplification products from the spleen, liver, ileum, and blood were confirmed as bovine V genes by cloning and sequencing. As shown in Fig. 8, an examination of five V sequences from the spleen showed a high level of diversity. Notably, in contrast to the fetal spleen, the liver, ileum, and blood yielded sequences that showed very little diversity (data not shown). These results demonstrate that only the fetal spleen shows significant V diversity at this stage of gestation. The regions of diversity in the fetal spleen sequences were compared with a pool of known germline V region genes (20 germline functional and pseudogene sequences from Parng et al. (19)) for possible donor sequences. The comparison implicated several possible donor sequences from the germline pool (see Fig. 9).

View larger version (51K): [in this window] [in a new window]   FIGURE 7. A 2% agarose gel with V PCR products from a variety of tissues in a 110 gestational day animal. PCR amplification of V genes from fetal tissues using primer sets specific for the V light chain region and for glyceraldehyde-3-phosphate dehydrogenase (GADPH), a common housekeeping gene, is shown.

View larger version (45K): [in this window] [in a new window]   FIGURE 8. Spleen sequences of bovine V cDNA from a 110 gestational day animal. The periods (.) indicate that the nucleotides were the same, the letters indicate the base-pair changes, and the asterisks indicate base deletions as compared with the root sequence. The root sequence was the most common sequence found in the fetal tissues.

View larger version (29K): [in this window] [in a new window]   FIGURE 9. Examples of GC in the spleen of a 110 gestational day animal. Donor sequences for GC were found in a pool of pseudogenes and germline genes. The functional germline gene R7 is compared with some of the expressed genes found in the fetal spleen. The potential germline or pseudogene donor sequence is also shown. Sequence differences that cannot be attributed to the donor sequence are italicized. The asterisk indicates a base-pair deletion.

	Discussion

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The functional significance of this early and extensive diversification has not been established; however, it is possible that the spleen in cattle serves a function similar to that assumed by the spleen in developing chickens. In the avian system, it is thought that B cells from the spleen seed the follicles of the bursa of Fabricius, where they subsequently undergo further diversification and clonal expansion (22). We speculate that in cattle, the fetal spleen may provide a partially diversified B cell stock from which B cells emigrate during early fetal life to colonize the developing follicles of the IPP. We further speculate that emigrant B cells in the IPP could undergo expansion and further diversify by somatic pm and GC.

The ability to isolate single IPP follicles and analyze clones from within those follicles allowed us to study the V diversity within the small and spatially isolated population of B cells in an individual follicle. Our studies of these B cell populations allowed us to assign a significant number of the sequences within a follicle to one or two genealogies. This is important for two reasons. First, the genealogic trees allow us to study the progenitor-progeny relationships within an individual follicle and determine how the sequences were derived from each other and, when possible, from a germline sequence. Tracing the successive descent of sequences from progenitor sequences has proven a useful tool for determining mechanisms of diversification (10, 20, 21). Second, the genealogies allow us to show that a progressive accumulation of mutations is occurring within one or more of the several clonal populations residing within an individual follicle. An examination of the trees we have constructed indicates that B cells are actively proliferating and mutating within the IPP follicle. We conclude that mutations accumulate and are passed from progenitor B cells to daughter B cells within the confines of an IPP follicle.

We compared the sequences within our genealogies with a pool of germline genes. Our hope was to find the suspected initial germline rearrangement that gave rise to the root sequences of the genealogies we traced. Although this could not be realized in every case, we were able to locate probable founder germline rearrangements for two of our three genealogies. We further compared the mutations in our genealogies with our pool of germline genes to try and find possible donor sequences for the mutations. Despite our lack of a complete set of germline genes, we were able to find donors for 58 of the 95 mutations observed in the individual IPP study. It is very difficult to determine whether a given mutation is a GC or a pm without a complete set of germline genes. Even with a complete set of germline genes, the certain attribution of isolated single base changes to GC cannot be completed with certainty, because there is a possibility that untemplated pm are actually responsible for base changes that appear to be templated.

We have also analyzed groups A, B, and C for the presence of commonly encountered murine somatic hypermutational "hot spots" (23). In mice, nucleotide motifs have been identified in which the usual random mutations associated with untemplated somatic hypermutation are observed to occur at a much higher frequency. Examination of our data showed that for groups A and C, few of the mutations (5% and 6%, respectively) correlated with hot spot motifs identified in mice. Such data suggest that no correlation exists between murine hot spots and mutations in groups A and C.

A similar hot spot analysis of group B determined that 53% of the mutations in this group were found in the context of a known murine hot spot motif. These data suggest that it may be possible to correlate some of the mutations found in cattle sequences with murine hot spot sequence motifs. The stark dichotomy between the groups does not allow us to draw any definitive conclusions about mouse hot spot sequence motifs and the mutations found in our bovine sequences. This analysis is subject to the caveat that uncertainty exists as to the extent that hot spot motifs identified in mice are the same as one might find in cattle.

In our analysis, many of the mutations identified as GCs involved apparent single base-pair changes. It is important to note that an indeterminate number of nucleotides 5' and 3' of the observed base change were probably involved in the GC event. However, these changes would remain unrecognized if they were identical with the founder sequence. In addition to the single changes, we found tracts of base changes present within some individual sequences in the follicles such as clone 44 from group A and clone 46 from group B. Our ability to template both single mutations and multiple tracts of mutations to an existing germline pool argues for the likelihood that GC is active in the IPP and contributes to Ig diversification in IPP follicles. Earlier work in the sheep by Reynaud et al. (7) applied a more stringent criterion for deciding whether mutations were GCs or untemplated pm. These workers only accepted the occurrence of tracts of mutations involving more than a single base-pair change. Imposing such stringent criteria has two likely outcomes. One is to give a greater level of confidence that the base changes attributed to GCs are actually the result of templated mutation. The other consequence of the use of such conservative criteria is to increase the likelihood that the contributions of GC to the observed diversification of a sequence will be underestimated.

Although we found potential templates for many of the mutations, there were 37 mutations for which we could not find donors within our pool of germline genes. We offer two possible explanations for these mutations. First, our inventory of germline genes is incomplete. Therefore, it is possible that many of the mutations could be templated by germline genes that we have not yet found and sequenced. Our second explanation is that both GC and untemplated somatic pm are active in the IPP follicle, and that all or a significant number of the single base changes we observe that are not traceable to a potential donor arise from untemplated pm. Evidence has been obtained in the rabbit (10) that is consistent with the operation of both GC and untemplated somatic pm. However, current methods of analysis would have no way to distinguish, unequivocally, between the two events. Therefore, we recognize the possibility that both GC and somatic pm may be operational in diversifying the primary repertoire in cattle.

The possible contributions of errors traceable to Taq polymerase must be considered in this analysis. The enzyme Taq polymerase is error prone and has a tendency to cause base-pair transitions of the A to G or T to C variety (24). To address this issue, an error measurement on a plasmid containing a fetal liver V gene was performed. Approximately 7,500 bp total were sequenced, and an error rate of 1 bp change in 2,517 bp (1/2517) was established. The errors observed were predominately the A to G transition usually associated with Taq error. Applying the error rate of (1/2517) to the 14,208 bp corresponding to the 48 sequences actually used to generate groups A and B (Figs. 1 and 2), we estimate that <6 of the 79 observed base-pair changes were due to Taq error. If five or six mutations were eliminated at random from groups A and B, the conclusions we have drawn would not change significantly.

In summary, this work establishes that in cattle, significant V diversification has occurred in the spleen before the initiation of diversification at follicular sites in the IPP. Genealogic analysis of libraries of V genes prepared from single IPP follicles demonstrates that follicles are actually a site of V diversification. Although other studies in sheep (25) and cattle (19) have established that IPP follicles and the IPP harbor highly diverse populations of V genes, this is, to our knowledge, the first demonstration that IPP follicles are environments in which mutations of V genes occur. However, since diversified populations of V genes are found in the early fetal spleen before the emergence of IPP follicles, there must be other sites where Ig genes can diversify at some point during developmental time. With respect to the mechanism of diversification, the examination of a large number of bovine V sequences confirms our earlier conclusion (19) that mutation is a major factor in V diversification in this species. Although careful examination of our data does reveal some examples of mutations that are most likely templated, there are many instances in which it is not possible to conclude, unequivocally, whether a particular pm arose from templated or untemplated mechanisms.

	Footnotes

 
¹ This work was supported by grants from the National Science Foundation (MCB-9405257) and the National Institutes of Health (GM36344 and AI37723).

² Address correspondence and reprint requests to Dr. Richard Goldsby, Life Sciences Building, Room 405, Amherst College, Amherst, MA 01002. E-mail address:

³ Abbreviations used in this paper: GC, gene conversion; pm, point mutation(s); IPP, ileal Peyer’s patch; LB, Luria-Bertani.

Accepted for publication July 9, 1998.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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