Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment

Cell, Vol. 49, 369478,

February

13, 1997, Copyright

0 1997 by Cell Press

Somatic Diversification of the Chicken lmmunoglobulin Light Chain Gene Is Limited to the Rearranged Variable Gene Segment Craig B. Thompson’ and Paul E. Neiman? Biochemistry Division lmmunobiology and Transplantation Department Naval Medical Research Institute Bethesda, Maryland 20814 t Fred Hutchinson Cancer Research Center Seattle, Washington 98104 l

Summary Previous studies have shown that the chicken 1 immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (V,,) and joining (Jn) coding regions. The immunologic repertoire of h genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabrlcius. This sequence dlverslficatlon was detected only at the rearranged V&, segment and not within the 5’ leader sequence, the Jk segment, or the unrearranged VII segment. The selective diversification of the rearranged Vbl segment was associated with unique DNAase l-hypersensitive sites on the rearranged allele. While probes for Vi1 sequences detect multiple homologous VI segments, plpbes for both the 5’ leader and J,, segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the li gene. Introduction Antibodies play a central role in the immune system because of their ability to recognize specific molecular antigens. In order to distinguish a wide variety of antigens, an organism must be able to generate a large number of different immunoglobulins. It has been estimated that mice and humans can generate between 106 and 10* different antibody molecules (Tonegawa, 1983; Teillaud el al., 1983). In these species, most of the differences between antibody molecules result from a series of somatic recombinations that occur during B cell differentiation and lead to the production of a functional immunoglobulin molecule (for review see Leder et al., 1980; Alt et al., 1986). In each B cell, a functional heavy chain gene is assembled from an assortment of variable (V), diversity (D), and joining (J) elements, and a functional light chain gene is assembled from an assortment of V and J sequences. These joining events themselves lead to the generation of further diversity through variations in the precise joining point and de novo nucleotide synthesis at the joining point (Weigert et al., 1980; Alt and Baltimore, 1982). A complete antibody molecule is assembled by combination of the rearranged heavy chain and the rearranged light chain, leading to a further amplification of diversity. While light

chains might be expected to have less diversity than the heavy chains because they lack D segments, additional light chain diversity is generated by having two independent genes (K and A), either of which can be recombined to form a functional immunoglobulin gene. Finally, further somatic diversification of both chains can be generated subsequent to rearrangement because of mutations, which appear to occur at higher rates within the immunoglobulin gene than elsewhere in the genome (Kim et al., 1981; Gearhart and Bogenhagen, 1983; Milner and Capra, 1983). It has been proposed that such mutations lead to clonal selection during the maturation of the immune response to a specific antigen (Griffiths et al., 1984; Clarke et al., 1985; Manser et al., 1985). Not all species, however, appear to use somatic recombination as a means of generating immunoglobulin diversity. The immunoglobulin heavy chain genes of the horned shark are organized in clusters containing single VH, D,+ JH, and CH segments (Hinds and Litman, 1986). This form of organization appears to limit the diversity created by combinatorial joining, and may play a role in restricting the range of the immune response in this lower vertebrate. In this species immunoglobulin diversity is created by the large number of individual heavy chain gene clusters that exist in the genome and can be used to generate a functional heavy chain. In contrast, the circulating immunoglobulins of the chicken have been shown to have a single light chain isotype (Grant 81 al., 1971). The chicken I immunoglobulin light chain locus has recently been characterized and has been found to contain a single Cl gene with a unique Jn element 1.9 kb upstream (Reynaud el al., 1983, 1985). By sequence homology to the variable region of a h cDNA, a cluster of 8-12 variable regions have been identified in the 20 kb upstream of the Jk segment. Despite this it was found that the same VA segment (V,) was joined to the J,, segment in most cells of the bursa of Fabricius. However, unlike the situation in elasmobranchs, the chicken does not have a restricted immune response in comparison with mammalian species (Grossi et al., 1976; Lydyard el al., 1976). In fact, chickens display considerable heterogeneity in their circulating immunoglobulin light chains as demonstrated by isoelectric focusing (Jalkanen el al., 1984). This suggests that chickens create diversity within their light chain genes by a process other than combinatorial joining. To date little is known about how somatic diversification of the chicken 1 gene occurs. While Reynaud et al. (1985) have shown by both sequence and restriction enzyme analysis that considerable sequence modification occurs at the VJ junction during VJ joining, they have also suggested that gene conversion and somatic mutation may play a role in the diversification of chicken immunoglobulin genes. To gain a better understanding of how somatic diversity develops within the 1 gene, we have examined the genomic organization of the VA, segment during B cell ontogeny. We confirm that the vast majority of bursal

Cell 370

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(A) Depicts the germ-line and rearranged h genes in bursal cells from SC chickens. DNA segments are shown as lines (flanking sequences and introns) and open boxes (exons). Pertinent restriction endonuclease sites are indicated, and the sequences corresponding to the U,, Us, and k probes are indicated by bars above the germ-line map. The two 1 alleles of these Fl birds differ at the two restriction sites marked by asterisks; both sites are present within one allele and are absent from the other. (B) DNA from bursal and thymic lymphocytes of a &week-old SC bird was digested with various combinations of restriction endonucleases as indicated above the lanes. Southern blots of these samples were hybridized with a nick-translated 1, probe. The resulting autoradiograms are shown, Lane M indicates end-labeled phage )c DNA digested with Hindlll and phage ax174 DNA digested with Haelll. The marker bands shown are, from top to bottom, 9.4, 6.6, 4.4, 2.3, 2.0, and 1.4 kb.

lymphocytes undergo a single rearrangement event that exclusively involves the Vxl segment. The genomic organization 5’ of the Vu segment is unaltered during both rearrangement and development. Restriction endonuclease sites within the rearranged, but not the unrearranged, VL1 gene are progressively lost during 6 cell development in the bursa of Fabricius. Cells that migrate from the bursa to the periphery are enriched for sequence alterations at these sites. In contrast, restriction endonuclease sites in the leader sequence 5’ of the Vu region and in the J1, region 3’ of the Vkl region are unaltered during rearrangement and development. Sursal lymphoma cells clonal for h rearrangements show heterogeneity with respect to the presence or absence of restriction endonuclease sites within the rearranged Vu region, demonstrating that somatic diversification can occur by events that occur subsequent to rearrangement. The selective diversification of the rearranged h gene is associated with both its transcriptional activation and the presence of DNAase l-hypersensitive sites flanking VL,. The selectivity of the diversification process for the Vh, segment suggests that somatic diversification could be the result of gene conversion between Vx, and the homologous Vh sequences located 5’ of VL1 or elsewhere in the genome. Such a process would spare the 5’ leader and JI segments since they lack homologous sequences in the genome.

Results Genomic Organization of the h Locus To analyze the events that occurred during and subsequent to 1 gene rearrangement, restriction endonuclease sites were mapped in both the rearranged and unrearranged alleles of bursal lymphocytes isolated from SC birds (Figure 1). These birds are the result of an Fl cross between two inbred chicken lines (see Experimental Procedures). The two a alleles in these Fl birds can be distinguished because of restriction-fragment length polymorphisms that exist between the two inbred parent strains at the SamHI endonuclease site 5’ of VA1 and at the EcoRl endonuclease site in the intron between JI and CL. These same polymorphisms have been observed in other strains (Weill et al., 1986). When bursal lymphocytes were isolated from 6week-old SC chicks and the DNA was analyzed for h gene rearrangements by Southern blotting, it was found that approximately half of the alleles had undergone the same 2.0 kb deletion. This deletion brought the V;* Seal site and the Jh Avrll site within 50 bp of each other. More than 95% of the bursal lymphocytes isolated expressed surface immunoglobulin, as determined by fluorescence-activated cell sorter analysis of the cells following staining with a murine monoclonal antibody specific for surface immmunoglobulin (data not shown). These data suggest that virtually all k rearrangements

Diversification 371

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Genes

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DNA was isolated from bursal (top panels) and splenic (middle panels) lymphocytes of SC chickens at various ages of development (from 15 days of embryogenesis to 16 weeks posthatching) and was digested with various combinations of restriction endonucleases as indicated. Southern blots of these samples were hybridized with a nick-translated I& probe, and equivalent exposures of the resulting autoradiograms are shown. The lower panels show schematic representations of the expected bands. In each case the germ-line band (G) results from a restriction site present within the DNA segment that has been deleted from the rearranged allele. The rearranged band(R) results from alleles that have undergone rearrangement but still maintain the germ-line Seal (S, left panels), Smal (S, middle panels), or Kpnl (K) sites within the rearranged VX1 segment. If the sequence of the rearranged Vkl segment has been modified at any of these sites, the length of the resulting band (M) will be determined by the next restriction site 5’ in VA,. The additional band seen at 4.4. kb in the Smal-Sal1 digests at some of the early developmental times results from partial methylation of the Smal site in the germ-line segment of contaminating nonlymphoid cells. Lanes M contain markers prepared as described in Figure 1. In the left-hand panels the 6.6, 4.4, 2.3, and 2.0 kb markers are shown; in the middle panels the 4.4, 2.3, and 2.0 kb markers are shown; and in the right hand panels only the 4.4 and 2.3 kb markers are shown

present in bursal lymphocytes must be functional, and that the majority of bursal lymphocytes possess one rearranged h gene and one germ-line 1 gene. Analysis of the polymorphic BamHl site demonstrated that rearrangement occurred randomly, and involved both alleles with approximately equal frequency as recently demonstrated by Weill et al. (1966). However, despite the fact that all bursal lymphocytes appear to have undergone the same rearrangement event, only 20% of the rearranged alleles have retained the Vu Seal site. The other 60% have lost the Seal restriction site either as a consequence of VJ joining or subsequent somatic diversification. Sequence Diversification within the Rearranged Vxl Segment during Development The genomic sequence of the unrearranged VA1 region contains several 6 bp restriction endonuclease sites (Reynaud et al., 1965). Since all bursal lymphocytes undergo the same rearrangement, we were able to test for sequence diversification of the Vht region by analyzing rear-

ranged h genes for the presence of restriction endonuclease sites found in the germ-line sequence of the Vhl region. Three restriction endonuclease sites within VA1 were chosen for these studies: Seal, Smal, and Kpnl. The Seal site is present at the 3’ end of the third complimentarity-determining region in V1,, just upstream of where VJ joining occurs. This region is thought to be involved in antibody recognition, and displays considerable sequence heterogeneity in mammals. In contrast, the Smal site is present within the framework region at the 5’ end of the variable segment. The framework region is thought to encode the structural backbone of the variable segment, and it shows a considerable degree of sequence conservation in mammals. The Kpnl site in Vhl is positioned at the junction between the first complementarity-determining region and the framework region and is between the Smal and the Seal sites in the Vhl segment. To test for diversification of the rearranged Vu segment during development, bursal and splenic lymphocytes

Cell 372

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DNA from bursal lymphocytes isolated from chickens at hatching (lanes H) and at 6 weeks of age (lanes SW) was digested with both Sail and the restriction endonuclease indicated at the top of each panel. Southern blots of these samples were hybridized with a nick-translated U, probe, and equivalent exposures of the resulting autoradiograms are shown. Shown below are schematic representations of the expected bands. In each case both the germline (G) and rearranged (R) alleles should yield the same band. Should sequence modification occur at one of the restriction sites within the rearranged VL1 segment, a new band would be created ending with the Sal1 site (M). These M bands do not hybridize with the U0 probe and are identical to the M bands identified in Figure 2 using the & probe, confirming that they are fragments containing the entire rearranged 1 locus. Seal, Smal, and Kpnl all have cleavage sites as indicated within the undeleted DNA segment. If sequence modification occurs within the unrearranged VH segment during development, new bands will appear that end at these sites; the expected positions of these bands are indicated by the arrows. Both the Smal and Kpnl digests also detect restriction sites 5’ of the ones described above. The 5’Smal fragment runs at about 6 kb and cannot be readily seen because of background hybridization of tJ1 to high molecular weight DNA. The 5’ Kpnl fragment is 900 bp and runs below the portion of the gel shown.

were isolated from birds at various ages between 15 days of embryogenesis, at which time the first cells expressing surface immunoglobulin can be detected in the bursa, and 6 months posthatching, when the bursa undergoes

involution. DNA was extracted from these cells, digested with Sall and either Seal, Smal, or Kpnl, and analyzed on Southern blots (Figure 2). All three sites were found to be progressively lost from the rearranged V1, segments of bursal lymphocytes during development. By 4 months of age, more than 90% of bursal lymphocytes were found to lack the Seal site, while more than 80% lacked the Smal and Kpnl sites. The rates of diversification of these three sites appear to be independent of each other, with the Seal site being lost most rapidly during development, followed by the Smal and then Kpnl sites. The B cell population that has migrated from the bursa to the spleen appears to be selectively enriched for sequence modifications at these sites. Somatic Diversification Does Not Occur in the Unrearranged Allele In contrast to the above results, we have been unable to detect loss of either the Seal or Kpnl sites in the Vhl segments of the unrearranged alleles, as shown by our failure to detect restriction fragments ending at the Seal or Kpnl sites within the undeleted DNA segment between Vhl and Jk on the unrearranged allele with a probe (U,) that comes from 5’ of Vkl (Figure 3). However, a strong band representing a restriction fragment ending within the undeleted segment of the germ-line allele was seen in all Smal digests analyzed with the lJ1 probe. The fact that this fragment spanned the VA, segment was confirmed by its hybridization with both a probe specific for leader sequences located just 5’ of Vkl and a probe specific for VA1 sequences 3’ of the V1, Smal site (see Figure 6). Thus it appeared that Smal failed to cut within the germline V;*, region at any time during development. This seems to be due to selective methylation of the germ-line Smal site in bursal lymphocytes, since this site could be cleaved in the germline VII segments of both red blood cells and thymocytes. In addition, we have redigested Smal-Sall digests with the methylation-insensitive isoschizomer of Smal, Xmal. This additional step leads to a significant reduction in the 2.1 kb band that results from failure of Smal to cut within the unrearranged VA, segment, but does not affect the intensity of the 4.1 kb band that results from failure of Smal to cut within the rearranged Vhl segment (data not shown). Somatic Diversification Can Occur Subsequent to Rearrangement Since diversification of the Vi1 segment was limited to the rearranged allele, it seems possible that diversification occurs exclusively during this process of rearrangement. Two pieces of evidence make this unlikely. First, the initial rearrangements detected at day 15 of embryogenesis retain the Vhl Seal site (Figure 4A), and mutations occur with time thereafter. This is consistent with a process that occurs not during initial h gene rearrangement but during the clonal expansion of B cells within the bursa subsequent to rearrangement. Second, avian leukosis virus (ALV)-induced bursal lymphomas that exhibit clonal h gene rearrangements can be polyclonal with respect to diversification at one or all of the VkI restriction endonu-

Diversification 373

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clease sites we have analyzed (Figure 4C). ALV induces a high frequency of bursal-dependent tumors in chickens. These tumors apparently arise as a result of ALV integration at or near the c-myc locus in bursal lymphocytes (Hayward et al., 1981). This integration event serves as a clonal marker for individual tumors (Neiman et al., 1980). We have analyzed two such ALV-induced bursal tumors. Both tumors exhibited clonal integration of ALV within a 14 kb fragment containing the c-myc locus (data not shown) and clonal rearrangement of one of their two h alleles (Figure 48). However, both tumors displayed a diversity of restriction endonuclease sites within the rearranged VX1. Since these tumors are clonal with respect to immunoglobulin gene rearrangement and ALV integration near c-myc, the only way for diversity to be created at the Seal, Smal, or Kpnl sites is for sequence modification to occur at these sites subsequent to rearrangement and tumorigenesis. In addition, tumor I is clonal with respect to diversification at the Seal site and is polyclonal at the Smal and Kpnl sites (Figure 4C). This suggests that diversification of the VA1 segment can be determined by multiple events separated in time. Neither tumor showed any significant loss of the Seal or Kpnl restriction sites in the unrearranged alleles (data not shown).

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(A) Initial rearrangements maintain the germ-line configuration of the VI, Seal site. Bursal DNAs from 15 day embryos (lane 15d) and newly hatched chicks (lane 4) were digested with both Seal and Salt Southern blots of these DNAs were probed with L and were exposed by autoradiography for 1 week. Positions for the germ-line (G), rearranged (R), and the modified rearranged(M) bands are indicated as described in Figure 2. Neither rearranged nor unrearranged alleles containing sequence alterations at the Seal site were detected. Similar results were obtained with Kpnl digests (results not shown). (B) lmmunoglobulin rearrangement and somatic diversification of the rearranged VH segment in bursal lymphomas. Two bursal lymphomas induced by clonal integration of ALV within the c-myc locus were examined for rearrangement of their I loci. Southern blots prepared following digestion of the tumor DNAs with BamHl and EcoRl were hybridized with the U, probe, and the resulting autoradiograms are shown. The liver DNA from the bird that had tumor II, and normal bursal DNA isolated from the uninvolved portion of the bursa from which tumor I was obtained were included as controls. This digestion scheme distinguishes between the germ-line (G) and rearranged (R) forms of both

Somatic Diversification Is Limited to Regions of h with Homologous Genomic Sequences At least two potential mechanisms exist for immunoglobulin diversification subsequent to rearrangement: point mutation and gene conversion. Two predictions concerning these possibilities can be made: First, if point mutations are occurring in a manner analogous to the way point mutations develop in mammalian immunoglobulin genes, they should occur throughout the rearranged k gene (Kim et al., 1981). Second, if gene conversion is occurring, it should be limited to sequences with homologous segments within the genome (Kourilsky, 1986). To test these possibilities, we next examined restriction endonuclease sites within the 5’ leader sequences and the J1 segment for evidence of somatic diversification similar to that occurring in the VX segment. Neither the 7 bp Mstll site present in the leader sequence nor the 6 bp Avrll site within the Jh segment shows any evidence of diversifica-

alleles because of restriction-fragment length polymorphisms for both restriction endonucleases (Figure 1). Positions of the potential bands Rr (7.4 kb), Gr (5.5 kb), Ga (5.1 kb), and As (4.6 kb) are indicated. Both tumors display clonal rearrangements of the allele containing the polymorphic BamHl and EcoRl sites. (C) Sequence modification of the rearranged V&r Seal, Smal, and Kpnl sites in bursal lymphomas. DNA from the bursal tumors described above was digested with Sal1 and either Seal (lanes 1 and 4) Smal (lanes 2 and 5) or Kpnl (lanes 3 and 6), and autoradiograms of Southern blots probed with ;lc are shown. (See Figure 2 for diagrammatic representations of potential restriction fragments.) While all of the rearranged VI, segments from cells of tumor I appear to have undergone sequence modification at the Seal site, each of the other digests demonstrated the simultaneous presence of cells with and without the respective restriction endonuclease site within Vi,. All three digests of tumor II revealed the simultaneous presence of cells with and without the respective restriction endonuclease sites within Vii.

Cdl 374

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(A) DNA from bursal lymphocytes isolated from chickens at hatching (lanes H) and at 6 weeks of age (lanes 6%‘) was digested with Bell. Half of each B&digested sample was then digested with Avrll. Southern blots of the four samples were hybridized with a nick-translated ;C, probe, and the resulting autoradiogram is shown above. Below is a schematic representation of the pertinent restriction sites. The Bell digests resulted in an 8.2 kb fragment from the rearranged allele and a 10.2 kb fragment from the germ-line allele. Following Avrll digestion of the Bell-restricted DNAs, a single 6.5 kb Avrll-Bell fragment was detected at both time points. No significant sequence modification of the Avrll site could be detected in the germ-line or the rearranged allele at either time point. (B) DNA from bursal lymphocytes isolated from chickens at hatching (lane H) and at 6 weeks of age (lane SW) was digested with Mstll. Southern blots of these samples were hybridized with a nick-translated Ur probe, and an exposure of the resulting autoradiogram is shown above. Below is a schematic representation of the expected bands. Both the germ-line (G) and rearranged (R) alleles should yield the same band. However, should sequence modification occur at the Mstll site within the rearranged allele, a new restriction band would be created (M). The 5’ Mstll fragment that is detected by the probe is indicated (5’frag.). Lane M contains markers prepared as described in Figure 1; the 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb bands are shown.

tion even though the Mstll site is only 175 bp upstream of the VL1 Smal site, and the Avrll site is only 35 bp downstream of the V&t Seal site (Figure 5). Other restriction endonuclease sites both upstream of Mstll and downstream of Avrll also failed to show any evidence of diversification, confirming these observations (results summarized in Figure 6). This sharp delineation of somatic diversification to the VxI segment was found to be associated with the presence of multiple homologous Vh segments within the genome, as described previously by Reynaud et al. (1985). In contrast, probes specific for the 5’ leader or the Jh segment failed to detect the presence of homologous sequences within the genome (Figure 6). The oligonucleotide probe specific for the 5’ leader sequence detected only the three Smal bands detected by a U, probe: the fragment ending at the VxI Smal site, the fragment ending at the Smal site between VL1 and Jh as a result of methylation of the germ-line Vk, Smal site, and the fragment resulting from loss of the Smal site in the rearranged Vi1 segment. A probe containing the Jk segment hybridized with the rearranged alleles that retained the Vhl Smal site and with the superimposed germ-line alleles and rearranged alleles that lack the Vxl Smal site.

The Rearranged VL1 Segment Is Defined by a Unique Chromatin Structure Potentially, the selective diversification of the rearranged V;I segment is determined by factors influencing higherorder chromatin structure. To test whether the chromatin structure surrounding the rearranged alleles was different from the chromatin structure surrounding the unrearranged alleles, the h alleles of both bursal and thymic lymphocytes were assayed for DNAase l-hypersensitive sites (Figure 7). In the bursal cells, the rearranged and unrearranged alleles were distinguished using a probe from the sequence of DNA deleted during rearrangement, which hybridizes only to the unrearranged allele (Figure 1). We found that both the rearranged and unrearranged I. alleles in bursal lymphocytes had chromatin structures distinct from those of the unrearranged alleles in thymic lymphocytes. The unrearranged allele had a major hypersensitive site within the DNA segment deleted during rearrangement that was not found in thymocytes. The rearranged allele had two unique hypersensitive sites flanking the Vbl segment, one occurring near the intron-5’ Vhl junction and one at the site of VJ joining. These hypersensitive sites were also present in DNA from bursal nuclei that

Diversification 375

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Discussion

Figure 6. Hybridization and Joining-Constant

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Variable

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(A) Southern blots of Smal digests of bursal DNA from &week-old chicks. Slots were probed with a 46mer oligonucleotide probe complementary to the V1, leader sequence (lane l), a 46-mer oligonucleotide probe complementary to an internal portion of the VH segment (lane 2) and a 2.7 kb DNA fragment containing the Jx and CL segments as well as the rntron sequences between them (lane 3). Equivalent exposures of the resulting autoradiograms are shown. The three bands in lane 1 are 6.4, 2.0, and 1.6 kb in length, and the same three bands also hybridize to the U, probe. The two bands in lane 3 are 6.4 and 4.7 kb, and both bands also hybridize to the kc probe. See the text for further description of the individual bands. (8) A schematic drawing of the rearranged 1 locus. The regions covered by the probes used in (A) are indicated by bars above the map. The positions of restriction endonuclease sites that have been analyzed for sequence diversification during development are shown. The sites found to undergo a high frequency of sequence modification during development are indicated with asterisks.

had undergone partial autodigestion by endogenous nucleases, suggesting that these sites might be potential sites of double-strand breaks in vivo (data not shown). In addition, the rearranged allele had a unique hypersensitive site 5’of the leader sequence, and two sites within the intron between Jh and Cl. The observed differences in the DNAase I hypersensitivity patterns between the rearranged and germ-line L alleles appeared to be associated not only with differences in the somatic diversification of the two alleles, but also with their transcriptional activity. We have been able to detect in bursal lymphocytes both a minor 4 kb and a major 1.8 kb L-associated transcript, which presumably represent the nuclear precursor and mature k messenger RNAs (data not shown). Both species fail to hybridize with probes from the segment that is deleted during rearrangement. Therefore, the differences in the chromatin structure of the unrearranged and rearranged alleles in bursal lymphocytes could be the result of differences in the transcriptional activity of the two alleles as suggested previously by Storb et al. (1986).

The chicken bursa of Fabricius provides an ideal opportunity to study the generation of antibody diversity during normal B cell development. Unlike other species, chickens do not appear to generate immunoglobulin light chain diversity as a result of somatic recombination. As reported previously (Reynaud et al., 1985) a single light chain gene rearrangement is detected in B cells derived from the bursa of Fabricius. This rearrangement results in joining of the Vhl segment to the J1 segment. While the choice of which 7, allele a cell rearranges appears to occur at random, the initial rearrangement event is nearly always functional. The vast majority of bursal lymphocytes therefore contain one rearranged and one germ-line allele. We have taken advantage of the unique opportunity provided by this situation to study the development of X light chain diversity during normal B cell development. By analyzing the rates of loss of restriction endonuclease sites present in both the rearranged and germ-line h genes we have gained a number of insights into how immunoglobulin diversity develops in a heterogeneous population of avian B cells. Using this approach, we were able to study events that were previously accessible to analysis only in cloned cell lines. Our results indicate that extensive sequence modification of the rearranged VII segment occurs during normal B cell development in the bursa of Fabricius. This process is not limited to sequences involved in VJ joining but can occur subsequent to VJ joining and lead to sequence diversity throughout the variable segment. The process of diversification is highly selective. It occurs in the rearranged VL segment but not in the unrearranged V1 segment. Protein coding sequences in both the leader and JL segments of the rearranged allele do not undergo detectable rates of modification. The diversification of the rearranged Vx segment within the bursa of Fabricius apparently leads to immunoglobulin sequence heterogeneity in mature B cells, since cells that have migrated from the bursa to the spleen appear to be selected for having undergone loss of germ-line V1, restriction endonuclease sites. Our data support the hypothesis that the bursa of Fabricius plays a unique role in the development of avian B cells (for reviews see Ratcliffe, 1985; Pink, 1986; Thompson et al., 1986a). The bursa first becomes populated with lymphoid stem cells between days 8 and 14 of embryogenesis. These cells begin to express surface immunoglobulin by day 12, and by the time of hatching at day 21 of embryogenesis nearly all (more than 95%) of bursal lymphocytes are surface-immunoglobulin-positive. After hatching of the chick, the bursa continues to increase in size as the number of cells in an individual follicle increases and B cells begin to seed to the other lymphoid organs. This process appears to involve clonal expansion of cells that have already rearranged their immunoglobulin genes rather than ongoing commitment of cells to the B cell lineage through immunoglobulin gene rearrangement (Weill et al., 1986). The bursa reaches its maximum size between 8 and 12 weeks of age and then begins to involute. By 6 months of age all that remains of

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5’ hypersensitive sites were analyzed on Southern blots prepared following Sal1 digestion of DNAs from a DNAase I digestion series of bursal and thymic lymphocyte nuclei prepared from the same bird (see Experimental Procedures). Blots were probed sequentially with ‘h, (left-hand panels) and Us (results not shown) to distinguish between sites within the rearranged and germ-line alleles. 3’ hypersensitive sites were analyzed on Southern blots prepared following Seal digestion of the same DNAs described above. Blots were probed sequentially with k, (right-hand panels) and U,, (results not shown). Positions of observed hypersensitive sites in the germ-line and rearranged h genes of bursal lymphocytes are depicted schematically below. One hypersensitive site (*) found within the germ-line allele of bursal lymphocytes was not found within the X loci of thymic cells. The 5’-most site and the two 3’-most sites map to the same regions on both the germ-line and rearranged alleles. The positions of all hypersensitive sites were confirmed using additional restriction digests and additional probes (results not shown). Lane M contains molecular weight markers prepared as described in Figure 1. The marker bands shown are 23.1. 9.4, 6.6, 4.4, 2.3, and 2.0 kb in length.

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LVJ

C

the bursa is a sclerotic remnant. Birds bursectomized at 17 days of embryogenesis fail to develop B cells and are agammaglobulinemic (Cooper et al., 1969), suggesting that normally B cell differentiation occurs exclusively in the bursa. In contrast, if the bursal epithelium is ablated at 60 hr of incubation, the resulting chickens lack a bursa but do develop circulating B cells and serum immunoglobulin (Eerola et al., 1984). However, these birds remain unable to mount specific antibody responses and are defective in their light chain diversity (Jalkanen et al., 1983, 1984). These data in conjunction with our results suggest that there is a specific mechanism activated during B cell development in the bursa of Fabricius that leads to the generation of antibody diversity in avian B cells. The data presented here do not indicate with certainty the mechanism by which somatic diversification of the rearranged h locus occurs within the bursa of Fabricius. To address this issue, Reynaud et al. (1987) have obtained sequence data from h cDNAs that demonstrate directly that gene conversion events are occurring between the V,, pseudogenes and the VLI segment. Several of our observations support their conclusion that gene conversion is the predominant mechanism by which chicken immunoglobulin light chain genes diversify. First, diversifica-

tion of restriction endonuclease sites is limited to the regions of the h gene that have homologous sequences within the genome (i.e., Vu and the VI pseudogenes). Second, sequence modification occurs at high frequency in regions of VLI that encode the framework portions of the light chain molecule. If these changes were the result of random mutations, one would expect that many mutations would lead to inactivation of the protein product and would be selected against. However, there appears to be little selection against loss of either the Smal or Kpnl sites of the Vi1 segments in peripheral B cells. This would be the expected result if sequence modification occurred at these sites as a result of gene conversion from alternative framework region coding sequences in homologous segments. Third, endogenous nuclease-hypersensitive sites exist at either end of the rearranged VL, segment, which might serve as regions of double-stranded breaks initiating homologous repair of the VL1 segment. Analysis of the molecular steps involved in the generation of Vi, sequence diversity should yield important insights into the mechanics of gene conversion. No matter what the precise molecular events are that allow for the high frequency of sequence modification within the rearranged Vi1 segment during bursal development,

Diversification 377

of Chicken

Light Chain

Genes

it is remarkable that we fail to detect any significant rate of diversification within the unrearranged V;I, segment. The simplest explanation for this observation is that sequence diversification requires an active gene conformation and/or VJ joining to initiate it. Sablitzky et al. (1985) have hypothesized that hypermutation of murine immunoglobulin genes is initiated by V segment rearrangement. Alternatively, diversification of the unrearranged allele may be actively suppressed. In mammalian species, transcription of the remaining germ-line variable segments appears to be suppressed following functional rearrangement (Yancopoulos and Alt, 1985). Potentially, mechanisms that regulate allelic exclusion may operate to suppress diversification as well (Storb et al., 1988). In support of this hypothesis, we found that there was selective methylation of the Smal site within the germline VL1 segment and that the chromatin structure of the deleted segment of the unrearranged h allele in bursal lymphocytes is different from that found in lymphocytes differentiating along the T cell pathway. Since the chicken h locus is so well defined, it should provide an ideal model for future study of how the germ-line allele is excluded from molecular events that occur with high frequency in the rearranged allele. Experimental

nick translation (for DNA fragments) or by elongation of a specific 16 mer primer using the Klenow fragment of DNA polymerase I (for oligonucleotide probes). Following hybridization, membranes were washed briefly at room temperature in 2x SSC, 0.1% SDS and then for 1 hr at either 56OC or 66°C in 0.1x SSC, 0.1% SDS. Membranes were then air-dried and then exposed to X-ray film for 12-48 hr at -70°C using intensifying screens. Band intensities were compared using densitometry as decribed previously (Thompson et al., 1986b). Analysis of DNAaee I-Hypereensltlve Sites Nuclei were isolated from thymic and bursal lymphocytes of Cweek-old chicks as described above. The nuclei were resuspended to a concentration of approximately 108/ml, and equal aliquots were exposed to increasing concentrations of DNAase I for 15 min. The digestions were stopped by adding an equal volume of a solution containing 1% SDS, 600 mM NaCI, 20 mM Tris (pH 7.4) and 5 mM EDTA. DNA was then isolated and analyzed as described above. Probes The U,, Us, and L probes used for these studies were derived from DNA fragments generously provided by C.-A. Reynaud and J.-C. Weill. The U, probe is a 1.4 kb Seal-Bgll fragment, the Us probe is a 1.3 kb Pvull fragment, and the hc fragment is a 1.2 kb EcoRI-Sall fragment (see Figure 1). The two synthetic oligonucleotide probes Land V were synthesized at the Fred Hutchinson Cancer Research Center. The L probe is a 46mer corresponding to the entire coding sequence of the leader sequence of V1, (Reynaud et al., 1985). The V probe is a 46mer corresponding to an internal fragment of the Vl, segment beginning 1 bp after the Smal site and extending in the B’direction (Reynaud et al., 1985).

Procedures Acknowledgments

Chickens The chickens used in these studies were Hyline SC birds, which are the result of an Fl cross between two inbred Sz chicken lines (Baba and Humphries, 1984). Fertile eggs (Hyline International) were incubated in a humidified incubator at 39OC until hatching. Thereafter the chickens were housed in the Animal Resource Center in accordance with National Institutes of Health guidelines. Bursal tumors were induced by injection of avian leukosis virus at either 10 days of embryogenesis or 1 day posthatching (Neiman et al., 1980). These animals were subsequently housed in isolation facilities and were sacrificed at 6-8 weeks of age. Cell lsolatlon and DNA Extraction Chickens were sacrificed at days 15 and 18 of embryogenesis. at day of hatching (day 21 of embryogenesis), and at 1, 2, 4, 6, 9, 16, and 24 weeks of age. At each time point the bursa and spleen were removed, a single-cell suspension was prepared, and the lymphoid cells from each organ were isolated following centrifugation of the cell suspension over Ficoll-Hypaque (Thompson et al., 1986b). Cell nuclei were isolated following cell lysis in RSB buffer (10 mM Tris [pH 7.41, 10 mM NaCI, 5 mM MgCl2) containing 0.5% NP-40. Nuclei were then solubilized in 0.5% sodium dodecyl sulfate (SDS), 300 mM NaCI, 10 mM Tris (pH 7.4) 5 mM EDTA. Proteins were digested with 250 &ml of proteinase K, and the DNA was precipitated in 2.5 M ammonium acetate and 50% (v/v) isopropanol. The precipitate was resuspended in TE (10 mM Tris [pH 7.41, 1 mM EDTA) and then reprecipitated as above. The resulting precipitate was recovered by centrifugation, washed twice with 70% ethanol containing 50 mM NaCI, and then resuspended to approximately 1 mg/ml in TE. Genomic Southern Blot Analysis Genomic DNA was digested with the appropriate restriction enzymes using conditions recommended by the supplier. DNA fragments were size-fractionated in 0.9% agarose gels and were transferred from the gel onto nitrocellulose membranes according to the method of Southern (1975). Membranes were baked under vacuum for 2 hr and then prehybridized at 42OC in a solution containing 50% formamide, 5x SSC, lx Denhardt’s solution, 25 mM sodium phosphate (pH 6.5) and 250 uglml Torula RNA. Hybridizations were carried out for 16-20 hr under identical conditions except for the addition of 10% dextran sulfate and 1 x lo6 cpmlml of the DNA probe. DNA probes were labeled by

We would like to thank Drs. C.-A. Reynaud and J.-C. Weill for many stimulating discussions and for their thoughtful review of the manuscript. We would like to thank Louise Carlson for her expert technical assistance, and our colleagues Peter Challoner, Harvey Eisen, Mark Groudine, David Bentley, Eric Humphries, and Michael Kuehl for their support and encouragement. These investigations were supported by National Cancer Institute grant CA 20068 and by the Naval Medical Research and Development Command, Research Task No. MR041.20.03.0002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

October

23, 1986; revised

December

9, 1986.

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