Switch region content of hybridomas: the two spleen cell Igh loci tend to rearrange to the same isotype

We have examined the switch region content of 25 hybridomas that secret antibodies of various isotypes with specificity for phosphocholine or glycoproteins of herpes simplex virus. These Southern hybridization experiments included probes for the murine JH region as well as probes for the mu, gamma 3, gamma 1, gamma 2b, gamma 2a, and alpha switch regions. For 22 of the hybridomas, the deletion model of the heavy chain switch fits the data well--all switch regions upstream of the rearranged (and expressed) switch regions are deleted and all switch regions downstream remain in the germline configuration. As exceptions to a simple deletion model of the switch recombination, we have observed two, and perhaps three, examples of switch region rearrangements downstream of an expressed heavy chain gene. The 25 hybridoma DNA samples include 28 rearranged gamma switch regions; the sizes of at least 25 of these rearranged fragments are consistent with recombination in the tandemly repeated sequences associated with gamma genes. For those hybridomas with two spleen cell-derived Igh loci, including three mu-expressers, three gamma 3-expressers, four gamma 1-expressers, and one gamma 2b-expresser, the two loci tend to be rearranged to the same switch region, suggesting that the heavy chain switch rearrangement is an isotype-specific event. The exceptions within this group include three hybridomas in which the switch seems to be incomplete--on one chromosome the JH complex is rearranged to the S gamma 3 region, while on the other it remains associated with the S mu region. A second group of hybridomas, which includes four gamma 3-expressers, have both gamma 3 and gamma 1 switch rearrangements. Each of these four hybridomas includes three rearranged JH segments, suggesting that they may be the result of an unusual differentiative pathway or a technical artifact. These experiments suggest that the heavy chain switch rearrangement in normal spleen cells is a deletion event that occurs within tandemly repeated elements. The rearrangement is mediated by factors with partial, or perhaps complete, isotype specificity.     

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.     

DNA sequences at immunoglobulin switch region recombination sites.

The immunoglobulin heavy chain switch from synthesis of IgM to IgG, IgA or IgE is mediated by a DNA recombination event. Recombination occurs within switch regions, 2-10 kb segments of DNA that lie upstream of heavy chain constant region genes. A compilation of DNA sequences at more than 150 recombination sites within heavy chain switch regions is presented. Switch recombination does not appear to occur by homologous recombination. An extensive search for a recognition motif failed to find such a sequence, implying that switch recombination is not a site-specific event. A model for switch recombination that involves illegitimate priming of one switch region on another, followed by error-prone DNA synthesis, is proposed.     

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.     

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.     

Isotype switching of an immunoglobulin heavy chain transgene occurs by DNA recombination between different chromosomes

Transgenic mice carrying an immunoglobulin mu heavy chain transgene exhibit isotype switching of the transgene. We have now characterized the mechanism of transgene switching in these mice. The site of mu transgene insertion in one transgenic line has been localized to chromosome 5 using a series of polymorphic endogenous retroviruses as genetic markers in backcross mice. The endogenous immunoglobulin heavy chain locus resides on mouse chromosome 12, which shows that transgene isotype switching can occur between two different chromosomes even though normal antibody gene switching has generally been thought to occur within one chromosome. We find that transgene isotype switching involves interchromosomal DNA recombination, and our data suggest that the same enzymatic mechanisms mediate both normal isotype switch recombination and interchromosomal transgene switching. Our findings also support the notion that the isotype switching mechanism can induce chromosomal translocations such as observed for the c-myc gene in some B cell tumors.     

Genomic distribution of heterochromatic sequences in equids: implications to rapid chromosomal evolution.

We describe a molecular model for rapid chromosomal evolution that proposes tandemly repeated DNA sequences as a driving force. A prediction of this model is that when extensive rearrangements of euchromatin have been facilitated by heterochromatin, genomes will be characterized by tandemly repeated sequences that have actively changed chromosomal fields by intragenomic movement. Alternatively, it is proposed that in conservative chromosomal lineage each class of tandemly repeated sequences will be restricted to a specific chromosomal field. To provide baseline data to test this model we examined four classes of tandemly repeated elements in six species of equids (Equus). Distribution of these sequences among species, as determined from slot blot analysis, and restriction site variation, shown by Southern blot hybridization, document that these sequences are in an evolutionarily dynamic state, and in situ hybridization documents extensive intragenomic movement among nonhomologous chromosomes and chromosomal fields. These data are interpreted as being compatible with the predictions of this model. Although this is clearly not the sole molecular factor driving chromosomal evolution, the model appears to be viable as an explanation of certain patterns of chromosomal evolution such as karyotypic megaevolution and some types of karyotypic orthoselection.     

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.     

Fish immunoglobulins and the genes that encode them

The nature of fish antibodies (concentrating primarily on the most studied species of bony and cartilaginous fishes) is discussed in terms of their immunoglobulin biochemistry and immunobiology. The major serum immunoglobulin (IgM) is described in detail, and structural variants of IgM are discussed in terms of their distribution in different fish species, and different anatomical sites within a fish (e.g. blood, mucus, bile). Structural variation in IgM includes the size of the constituent heavy and light polypeptide chains, and the extent to which they are covalently associated with one another. The intramolecular heterogeneity of binding sites for antigen on IgM is reviewed and possible mechanisms for the phenomenon are presented. The evidence suggests that some, but not all, species of fish possess a detectable J chain in their IgM. The general nature of the fish immune response is that IgM antibody of moderate affinity is produced and prolonged or repeated immunization: (a) fails to produce a switch to production of a non-IgM class of antibody, and (b) fails to induce substantial increases in the affinity of the specific antibodies. Evidence supports a conclusion that fish lack the typical secondary antibody response seen in mammals, and possess antibodies of limited heterogeneity. Our current understanding of the genetic basis for fish antibodies is presented. Fish appear to utilize the same basic genetic elements as mammals to encode and regulate the expression of their immunoglobulins. The teleost heavy chain (IgH) locus resembles that of mammals and amphibia in its organization. The IgH locus of elasmobranchs is arranged in a unique multicluster organization. The light chain loci of elasmobranchs are organized analogously to the heavy chain locus (in multiclusters). The structure of the light chain locus of teleosts is presently unknown. Teleost fish utilize a unique pattern of RNA processing to generate the secreted and membrane receptor forms of the IgM heavy chain. The genes encoding the unique low molecular weight Ig heavy chain found in skates and rays have been cloned and sequenced, and also display the multicluster pattern of organization. Teleost fish appear to have normal numbers of variable regions: it is hypothesized (but as yet unproven) that the failure of their IgM to increase in affinity is due to a deficiency of somatic hypermutational mechanisms in their Ig gene variable regions during B lymphocyte differentiation.     

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.     

DNA sequence of the murine gamma 1 switch segment reveals novel structural elements

Immunoglobulin heavy chain switch regions are segments of DNA considered to be important in mediating class switching in B lymphocytes. Whereas these segments vary in length among the different murine isotypes, their structural organization schemes are all based on the tandem repetition of unit sequences. We previously showed that the S gamma 1 segment unexpectedly contains sequence elements that differ significantly from its prevalent unit repeat (49mer). Here we extend this preliminary characterization by determining the complete nucleotide sequence of the cloned S gamma 1 segment from BALB/c DNA. We find that S gamma 1 consists of more than 120 tandemly repeated 49mers. In addition, we show that the previously identified non-49mer sequences are part of a direct repeat element about 350 bp in length (DR II), which exists in two copies at the 5' end of S gamma 1. We also show that another unrelated direct repeat element about 500 bp long (DR I) exists near the 5' and 3' ends of S gamma 1. Thus, the structure of the S gamma 1 segment might be may be abbreviated as 5'-DRII-(49mer)15-DRI-DRII-(49mer)n-DRI , where n is between 40 and 160. Our results of Southern hybridization experiments suggest that this basic structural scheme is maintained in eight different Igh haplotypes, although S gamma 1 segments in different Igh haplotypes include different numbers of 49mer elements. Other murine S gamma segments differ in size among various Igh loci, but to a lesser extent than S gamma 1. At the level of tandemly repeated sequences, S gamma 1, S gamma 3, and S gamma 2b represent three distinct, nonoverlapping sets of sequences.     

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.     

Nuclear matrix attachment occurs in several regions of the IgH locus

The genome is thought to be divided into domains by DNA elements which mediate anchorage of chromosomal DNA to the nuclear matrix or chromosome scaffold. The positions of nuclear matrix anchorage regions (MARs) have been mapped within the 200 kb mouse immunoglobulin heavy chain constant region locus, thereby allowing an estimate of the size of DNA domains within a segment of the genome. MARs were identified in four regions, which appear to divide the locus into looped DNA domains of 30, 20, 30 and greater than 70 kb in length. These DNA domain sizes fall within the range of DNA loop sizes observed in histone-extracted nuclei and chromosomes. In two regions, large clusters of MARs were identified, and many of these MARs lie on DNA fragments that include repetitive DNA elements, perhaps indicating that repetitive DNA integrates into the genome close to MARs, or that some classes of repeats could themselves act as MARs.     

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.     

Site-specific recombination between immunoglobulin D and JH segments that were introduced into the genome of a murine pre-B cell line

A recombinant plasmid containing the herpes simplex virus thymidine kinase (tk) gene, flanked on one side by two murine immunoglobulin heavy chain diversity (D) elements and on the other by two murine immunoglobulin heavy chain joining (JH) elements, was introduced into a tk- variant of a pre-B cell line transformed by Abelson murine leukemia virus. The four possible site-specific joining events between the D and JH segments within the integrated construct occurred frequently during passage of the cloned line under nonselective conditions, and deletion of the internal tk gene as a result of these joining events was, by far, the predominant mechanism of resistance to BUdR within this line. These studies demonstrate that a precise chromosomal location is not essential for the assembly of D and JH elements and provide a model system for mechanistic and genetic studies of this recombination process.     

Preferential integration of the Ad5/SV40 hybrid virus at the highly recombinogenic human chromosomal site 1p36

Human fibroblasts transformed with an adenovirus-5/simian virus 40 recombinant construct (Ad5/SV40) were analyzed to determine the chromosomal site(s) of virus integration. This was firstly done by in situ hybridization using metaphase and prometaphase chromosomes and 125I-labeled Ad5 DNA. Out of seven transformed cell lines (six of clonal origin and one uncloned), six were proven to have integrated the viral genome at the short- or the long-subtelomeric regions of autosome 1, two regions known to include chromosomal modification sites induced by acute infection with Ad12. Characterization of the integration sites was carried out by restriction analysis. Transformed cell lines with the same major chromosomal integration site were found to have the viral genome inserted in restriction fragments of different size, indicating that viral integration has occurred at different sites within a relatively small chromosomal region. Molecular studies carried out on one of the transformed cell lines (H13.1) gave an independent confirmation of the viral integration at the subterminal region of autosome 1 short arm. Nucleotide sequencing at this cellular-viral junction has shown that the virus has integrated within tandemly repeated Alu-like elements and that the cellular flanking sequences have several homologies with variable number of tandem repeats core sequences. Many possible open reading frames were identified in the DNA segment adjacent to the Alu-like elements.     

Organization of variable region segments of the human immunoglobulin heavy chain: duplication of the D5 cluster within the locus and interchromosomal translocation of variable region segments.

We have studied the organization of variable region (V) genes of the human immunoglobulin heavy chain (H) by cosmid cloning. We isolated two independent immunoglobulin D5 clusters (D5-a and D5-b) from cosmid libraries of the human genome. Restriction maps of these two regions showed that downstream 15 kb portions of the 55 kb overlap were different although upstream 40 kb portions were almost identical. Four more D segments, (DM, DXP, DA and DK) were found around the D5 segment in the conserved region of each cluster. Nucleotide sequences of the corresponding D segments from each cluster were almost identical and they encoded potentially functional D regions. Analysis using human-rodent somatic cell hybrids demonstrated that both clusters were located in the immunoglobulin heavy chain (H) locus on chromosome 14, suggesting that the D5-a and D5-b regions evolved by internal duplication within this locus. We also isolated a 60 kb DNA region carrying four VH segments, designated as VH-F region, which was located on chromosome 16. Nucleotide sequences of the four VH segments were determined. Two of them encoded potentially functional VH segments, and the other two were pseudogenes. Some more VH segments were found to be located outside chromosome 14, by Southern blot hybridization of human-rodent hybrid cell DNAs. These results provide further evidence that the human VH locus has undergone recent reorganization.     

The immunoglobulin heavy chain class switch

Conclusions While much has been learned concerning the molecular structural basis for the heavy chain class switch, many questions relating to the regulation of the switch remain unanswered, or at least controversial. Identification of the enzyme system which mediates the class switch, as well as other regulatory, possibly X-linked, genes should provide the necessary key to our understanding of this unique process.AbbreviationsB cell lymphocyte derived from the bone marrow in adult mammals or the bursa of Fabricius in chickens - bp base pair - C immunoglobulin constant region - CDR complementarity-determining region of the immunoglobulin variable region - D diversity gene segment of the immunoglobulin heavy chain variable region gene - H immunoglobulin heavy chain - Ig immunoglobulin - J joining region gene segment of the immunoglobulin variable region gene - kb kilobase - L immunoglobulin light chain - LPS lipopolysaccharide - Pyr pyrimidine - S-, s-site, s-region switch rearrangement site - SCE sister chromatid exchange - sIg surface immunoglobulin - T cell lymphocyte derived from the thymus - USCE unequal sister chromatid exchange - V immunoglobulin variable region