The DJH complex remains active in recombination to VH segments after the loss of mu-chain expression in mu-positive pre-B cells

AT11-2 is an Abelson virus-transformed B precursor cell line which is capable of differentiating Ig- from mu+ cells via functional recombination of VH segments to preexisting DJH complexes. We describe here that after a mu+ subclone (VDJ+/DJ) generated from Ig- AT11-2 (DJ/DJ) cells by in vitro functional VH to DJH recombination subsequently lost mu-chain expression either by the recombination of a pseudo VH segment to the VHDJH+ allele or by the deletion of VHDJH+ allele, a novel productive joining of VH segments to the preexisting DJH complex occurred. These results indicated that VH to VHDJH rearrangement was not suppressed in mu-chain producing cells and that the DJH complexes still remained active in the recombination to VH segments after the loss of mu-chain expression. Our results may also suggest that VH to DJH rearrangement, but not VH to VHDJH rearrangement, is suppressed in mu-chain producing cells to maintain allelic exclusion. Our cell differentiation system should continue to be valuable for elucidating the mechanism of suppression and associated implications regarding allelic exclusion.     

Recombination events near the immunoglobulin Cmu gene join variable and constant region genes, switch heavy-chain expression, or inactivate the locus

Immunoglobulin heavy-chain expression is initiated by recombination between a variable region (VH) gene and one of several joining region (JH) genes located near the mu constant region (Cmu) gene, and the active VH gene can subsequently switch to another CH gene. That the general mechanism for CH switching involves recombination between sites within the JH-Cmu intervening sequence and the 5' flanking region of another CH gene is supported here by Southern blot hybridization analysis of eight IgG- and IgA-secreting plasmacytomas. An alternative model requiring successive VH linkage to similar JH clusters near each CH gene is shown to be very unlikely since the mouse genome appears to contain only one complement of the JH locus and no JH gene was detectable within large cloned sequences flanking germline C gamma 3 and C gamma 1 genes. Thus, VH-JH joining and CH switching are mediated by separate regions of "the joining-switch" or J-S element. In each plasmacytoma examined, the J-S element had undergone recombination within both the JH locus and the switch region and was shown to be linked to the functional CH gene in an IgG3, and IgG1, and three IgA secretors. Both JH joining and CH switching occurred by deletion of DNA. Switch recombination occurred at more than one site within the J-S element in different lines, even for recombination with the same CH gene. Significantly, although heavy-chain expression is restricted to one allele ("allelic exclusion"), all rearranged in each plasmacytoma. Some rearrangements were aberrant, involving, for example, deletion of all JH genes from the allele. Hence, an error-prone recombination machinery may account for allelic exclusion in many plasmacytomas.     

VHDJH formation and DJH replacement during pre-B differentiation: non-random usage of gene segments.

The Abelson murine leukemia virus (A-MuLV) transformed cell line 300-19 was derived from the bone marrow of an adult NIH/Swiss outbred mouse. The original 300-19 clonal isolate carried DHH rearrangements of both JH alleles, a molecular genotype characteristic of early pre-B cells. During propagation in culture, the 300-19 line frequently generates secondary rearrangements of its JH alleles including rearrangements which append VH segments to the pre-existing DJH complexes to form complete VHDJH variable region genes and secondary D to JH rearrangements which replace the pre-existing DJH rearrangement by joining an upstream D to a downstream JH. The two types of secondary rearrangement events occur at approximately equal frequency. Approximately 30% of the VH to DJH joins lead to the production of mu heavy chains providing support for a regulated model of allelic exclusion. Like pre-B cell lines from other origins, the 300-19 line preferentially utilized VH gene segments from the more JH-proximal (3') families to form VHDJH rearrangements. However, the VH segments preferentially employed by 300-19 were from a different family than those previously demonstrated to be utilized by pre-B lines of BALB/c origin; we relate these different utilization patterns to differences in the organization of the more 3' VH families between the two strains. The initial DJH rearrangements of the 300-19 line employed more 3' (JH-proximal) D segments; however, the DJH replacements preferentially employed the most 5' D segment. We discuss this phenomenon in the context of a mechanism which may target recombinase to regions of the chromosome more 5' to the D locus (VH-containing regions) once an initial DJH complex is formed.     

Ordered rearrangement of immunoglobulin heavy chain variable region segments

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.     

Immature B cells can pass through a VHDJH/germ line state in the Ig H chain gene rearrangements

SPL2-1-2, an Ig- murine immature B cell line, was established by the infection of immature B cells with tsOS-59, a temperature-sensitive mutant of Abelson murine leukemia virus. Southern blot and DNA cloning and sequencing analysis showed that SPL2-1-2 had a nonproductive VHQ52.DSP2.JH3 and a germ line (G) allele (VHDJH-/G), and that D to JH joinings followed by VH to DJH joinings progressed on the G allele during culture. These results indicated the existence of novel pathway of IgH gene rearrangements: G/G----DJH/G----VHDJH/G----VHDJH/DJH----VHDJH /VHDJH. This also implied that whether DJH/G state progressed to DJH/DJH or VHDJH/G state was stochastically determined, but not strictly controlled in an ordered fashion, although DJH/G state progressed more preferentially to DJH/DJH state than to VHDJH/G state.     

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.     

Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.     

Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.     

Evidence that terminal deoxynucleotidyltransferase expression plays a role in Ig heavy chain gene segment utilization

TdT is a nuclear enzyme that catalyzes the addition of random nucleotides at Ig and TCR V(D)J junctions. In this paper we analyze human IgH rearrangements generated from transgenic minilocus mice in the presence or absence of TdT. In the absence of TdT, the pseudo-VH gene segment present in the minilocus is rearranged dramatically more frequently. Additionally, JH6 gene segment utilization is increased as well as the number of rearrangements involving only VH and JH gene segments. Thus, the recombination of IgH gene segments that are flanked by 23-nt spacer recombination signal sequences may be influenced by TdT expression. Extensive analysis indicates that these changes are independent of antigenic selection and cannot be explained by homology-mediated recombination. Thus, the role played by TdT may be more extensive than previously thought.     

Isolation of an IgH gene circular DNA clone from human bone marrow.

Circular DNA was obtained from human bone marrow. Then a phage library was prepared and screened by use of two probes of the IgH gene; 5'-DHQ52, containing the 5' flanking region of DHQ52, and JH4.3, containing the sequence from JH3 to the 3' flanking region of JH6. One clone, HBMC-1, that was DHQ52+JH4.3- was obtained. HBMC-1 had the germline IgH region upstream of JH1 and the 3' flanking region of DXP1. A recombination signal sequence flanking the 5' side of the JH1 segment was attached to the recombination signal sequence flanking the 3' side of DXP1 forming a head-to-head structure of two 7mers with 10 nucleotides in-between. HBMC-1 is thus considered to be a circular DNA deleted as a consequence of DXP1-JH1 joining of the IgH gene.     

Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation.

Sequence analysis of rearranged VHDJH genes of B lineage cells from various stages of ontogeny indicates that short sequence homologies at the breakpoints of recombination contribute to V region gene assembly. Such homologies are regularly seen at DJH junctions of neonatal pre-B cells, most of which do not contain N sequences. In the same cells, but not at later developmental stages, preferential usage of the JH1 element is observed. After birth, N sequence insertion increases with time and is always more prominent at the VHD border than the DJH border. In pre-B cells from adult animals and in mature B cells, in cases where N sequences were not detectable, sequence homologies at the DJH border were found in only half of the instances. This lower incidence could be due to N sequence addition to one of the recombining DNA ends and/or cellular selection. Inspection of VHDJH junctions for N sequence insertion, sequence homologies at the DJH border and JH1 usage allows the estimation of the timepoint in ontogeny at which particular B cell subsets are seeded into the immune system. Specifically, the present data show that the cells of the Ly1 B cell subset are generated not only neonatally but also beyond the first weeks of life. However, the DJH junctions of the progenitors of chronic B cell leukemias which originate from the same B cell subset resemble those of neonatal pre-B cells, suggesting that these cells have already undergone a transforming event at this early developmental stage.     

The use of chromosomal translocations to study human immunoglobulin gene organization: mapping DH segments within 35 kb of the C mu gene and identification of a new DH locus.

We have studied the Burkitt's lymphoma cell line Daudi which carries the translocation t(8;14). The breakpoint of this translocation on the 14q+ chromosome occurs near to a rearranged DH-JH join, and the actual chromosome junction is a few hundred base pairs upstream of the joined DH element. The nucleotide sequence of the rearranged DH segment shows that it does not come from the previously described D cluster. Using this DH sequence as a probe we have identified two separate DH clusters. One of these is the major DH cluster and is located only 20 kb upstream of the JH segments. A pseudo-VH (probably the first VH segment) is also found approximately 98 kb from JH. A second, minor DH locus has been found which seems to be located on the distal side of the VH locus on chromosome 14, since there is little evidence for rearrangement or deletion of this locus in any B cell DNA analysed. A single VHIII subgroup gene is located within 25 kb of the newly identified DH element: it is possible that this minor locus occurs near the limit of the Igh locus.     

Immunoglobulin V region variants in hybridoma cells. II. Recombination between V genes.

The mouse hybridoma line B1-8.delta 1 secretes a monoclonal IgD, lambda 1 anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V-region variants (B1-8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1-8. delta 1 and variant heavy chain V regions were determined. The variant VH regions are identical. Wild-type and variant VH regions differ in 10 positions. Single amino acid exchanges are found in the first and second framework at positions 20 and 43. The majority of replacements (eight substitutions) is clustered in the second complementary-determining region (CDR 2). There are no differences in CDR 1 and CRD 3 and the JH region. The variant, which at first glance appears to have undergone a series of point mutations, arose by recombination, possibly gene conversion, between the rearranged VDJ gene of the wild-type (B1-8.delta 1) and a neighbouring germ line VH gene encoding all of the substitutions.     

Immunoglobulin V gene replacement is caused by the intramolecular DNA deletion mechanism.

Circular DNA resulting from V gene replacement was studied with an A-MuLV transformed cell line containing ablts. This cell line undergoes V gene replacement at elevated temperatures in the immunoglobulin (Ig) heavy chain (H) gene. Examination of circular DNA revealed that a heptamer-related sequence (TACTGTG) within the coding region of VDJ was joined to the recombination signal sequence (RSS) of a germline VH segment. This provides direct evidence for a intramolecular DNA deletion mechanism for V gene replacement. In the pre-B cell line as well as in in vivo lymphocytes, unusual circular DNAs were found which were structurally similar to the V gene replacement circles. They represented excision products of the deletion type recombination between one complete RSS and a heptamer-like sequence in the Ig H region.     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

Members of novel VH gene families are found in VDJ regions of polyclonally activated B-lymphocytes.

Four potentially productive and two non-productive VDJ gene segments were isolated from the DNA of mouse B-lymphocytes which had been polyclonally activated by bacterial lipopolysaccharide (LPS). Three VDJ regions exhibit VH genes which stem from two novel VH gene families. The complexity of these families is 5-9 genes. One of the non-productive VDJ regions exhibits a D segment which may have been generated by joining of two DSP2 segments. Both non-productive VDJ regions appear to contain rearranged pseudo VH genes. Three potential somatic mutations distributed over two productive VDJ regions are observed.     

Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice

Studies of Ig and TCR genes in transformed lymphocytes of scid mice have revealed aberrant DNA rearrangements. Here we present a more detailed analysis of the Igh gene recombination in nine scid pre-B cell lines transformed by Abelson murine leukemia virus. We found 85% of the rearranged Igh alleles to contain abnormal Dh-Jh deletions of varying size. All of these deletions encompassed Jh elements and extended into the Igh enhancer region, occasionally involving the switch (S) region of the C mu gene. Some of these rearrangements removed most of the Dh elements, but none appeared to extend to the Vh genes. DNA sequence analysis of the two abnormally rearranged Igh alleles in one pre-B cell line showed that no Dh or Jh coding sequences were retained at the recombination sites though heptamer-like (CACTGTG) recognition signal sequences were present in the absence of nonamer (GGTTTTTGT) recognition signal sequences. These results imply that a deregulated recombinase activity may be responsible for the abnormal Dh-Jh deletions and the absence of Vh-Dh joining in established lines of Abelson murine leukemia virus-transformed scid pre-B cells.