Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch.

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

Complete nucleotide sequence of the murine gamma 3 switch region and analysis of switch recombination sites in two gamma 3-expressing hybridomas

The heavy chain isotype switch is mediated by a DNA rearrangement between a donor switch region (usually mu) and a recipient switch region (gamma, epsilon, or alpha). Switch regions lie upstream of the appropriate heavy chain constant region gene and are composed of simple sequences repeated in tandem. It is not known to what extent the tandemly repeated sequences are important to the heavy chain switch recombination, and to what extent other features of switch region sequences might contribute to the switch process. We studied switches to the gamma 3 isotype by sequencing the entire gamma 3 switch region. This switch region is composed of forty-four 49 base pair units repeated in tandem. These repeated units share modest homology with the mu switch region repeated elements. Evolution of the gamma 3 switch region seems to involve insertions and deletions of the 49mer elements. We also molecularly cloned rearranged switch regions from two gamma 3-expressing hybridomas and determined the DNA sequences at the mu-gamma 3 recombination sites. We located these switch recombination sites within the germ-line gamma 3 switch region, as well as switch recombination sites from two myelomas. All four sites are found in the 5' one-third of the gamma 3 switch region. We discuss some additional trends in the sequence data near these four recombination sites.     

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.     

Circular DNA is excised by immunoglobulin class switch recombination

We have purified extrachromosomal circular DNAs from adult mouse spleen cells, and cloned into a phage vector the BamHl fragments hybridizing with C mu and S gamma 1 probes. We obtained 52 S mu+S gamma 1+ clones by screening 1.4 million phage clones derived from spleen cells stimulated with bacterial lipopolysaccharide and interleukin 4. We have identified the breakpoints of six clones that contain S gamma 1 and S mu sequences fused in the 5' to 3' orientation. All these switch recombination sites were assigned to the central repetitive sequences of the S mu and S gamma 1 regions. Since the common S mu-S gamma 1 sequences at the recombination sites are at most 2 bases long, typical homologous recombination cannot account for their joining. These findings provide direct evidence that mu-gamma 1 class switching can occur by the looping out and excision of chromosomal DNA, with formation of a circle.     

Isolation and characterization of endonuclease J: a sequence-specific endonuclease cleaving immunoglobulin genes.

An endonuclease activity which cleaves close to the recombination sites of the immunoglobulin JK segments was found in extracts of chicken bursa of Fabricius and characterized after partial purification. The enzyme preparation also cleaved a VK segment at its 3' end. A similar activity was found in mouse liver, mouse myelomas and Hela cells. The enzyme designated as endonuclease J introduces double-stranded cleavages preferentially at sequences containing G clusters of pBR322 as well as the JK segments. However, not all the G clusters were cleaved by endonuclease J, suggesting that the enzyme recognizes additional sequences. Deletion of the conserved nonamer (GGTTTTTGT) located immediately 5' to the JK4 segment drastically reduced the cleavage activity of its immediate downstream G cluster. Although biological function of endonuclease J is not clear at this stage, the possibilities of its involvement in the immunoglobulin gene recombination and general recombination were discussed.     

Rearrangement of rat immunoglobulin E heavy-chain and c-myc genes in the B-cell immunocytoma IR162.

Mammalian B-cell lymphoid malignancies frequently display aberrant translocations involving the c-myc proto-oncogene and one of the immunoglobulin loci. We have observed and characterized such a translocation in the immunoglobulin E-producing rat immunocytoma IR162 by using recombinant DNA technology. We show here for the first time that a c-myc gene has recombined with the excluded allele of the nonfunctional epsilon heavy-chain immunoglobulin gene. This recombination has resulted in the loss of 5'-proximal DNA and, consequently, potential regulatory information from the body of the c-myc structural gene via joining to the epsilon heavy-chain switch region in a head-to-head, i.e., 5'-to-5', configuration. As discussed in this report, these results, together with the previous results of others, have important implications for immunoglobulin heavy-chain class switching mechanisms controlling normal and abnormal translocations.     

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.     

(AGCTG) (AGCTG) (AGCTG) (GGGTG) as the primordial sequence of intergenic spacers: the role in immunoglobulin class switch

The means employed for immunoglobulin heavy chain class switch appears to be no different from that by which meiotic intergenic crossing-overs at accomplished. As with other intergenic spacers, the 5' noncoding sequence of each Ig CH (immunoglobulin heavy chain constant region) gene apparently undergoes unconstrained sequence changes due to randomly sustained base substitutions, deletions, and duplications. Yet, there remains sufficient regional sequence homology between the Ig Cmu 5' noncoding sequence and those of its somatic recombination partners, e.g., Ig C gamma 1, Ig C gamma 2b, Ig C alpha, because each of these 5' noncoding sequences is made of multiple copies in various stages of degeneracy of one primordial 20 base pair-long sequence: (AGCTG) (AGCTG) (AGCTG) (GGGTG).